SiRNA-based cancer treatment

ABSTRACT

A double-strand oligonucleotide including two complementary oligonucleotide sequences forming a hybrid, each including at one of their 3′ or 5′ ends, one to five unpaired nucleotides forming single-strand ends extending beyond the hybrid, one of the oligonucleotide sequences being substantially complementary to a target sequence belonging to a DNA or RNA molecule to be specifically repressed, the target sequence belonging to a DNA or RNA molecule of a gene coding an angiogenic factor.

RELATED APPLICATIONS

This is a divisional of U.S. Ser. No. 10/494,800, filed May 6, 2004, which is a §371 of International Application No. PCT/FR02/03843, with an international filing date of Nov. 8, 2002 (WO 2003/040366, published May 15, 2003), which claims priority of French Patent Application Nos. FR 01/14549, filed Nov. 9, 2001, and FR 02/04474, filed Apr. 10, 2002.

TECHNICAL FIELD

This disclosure relates to the field of the genetic investigation and treatment of human pathologies, especially cancers and infectious diseases.

BACKGROUND

Known in the prior art are antisense oligonucleotide techniques making it possible to specifically inhibit a gene in mammal cells. These techniques are based on the introduction into cells a short oligonucleotide of DNA that is complementary to the target gene. This oligonucleotide induces the degradation of the messenger RNA (mRNA) transcribed by the target gene. Another antisense technique comprises introducing into a cell a DNA oligonucleotide which forms a triple strand with the target gene. The formation of this triple strand represses the gene by either blocking access for activating proteins, or in more sophisticated approaches, by inducing degradation of the gene. None of these approaches appear to be based on a cellular mechanism existing in the cells of mammals, and they are not very effective. In fact, the clinical use of antisense has been reduced to a few rare cases, and it was believed that there was no possible use for oligonucleotides forming triple strands.

Interference RNA which is also designated “RNA'inh” or “RNAi” or cosuppression, has been demonstrated in plants. It was observed in plants that the introduction of a long double-strand RNA corresponding to a gene induced the specific and effective repression of the target gene. The mechanism of this interference comprises the degradation of the double-strand RNA into short oligonucleotide duplexes of 20 to 22 nucleotides.

The “RNA'inh” approach, more generally referred to according to the invention as inhibitory oligonucleotides or RNAi, is based on a cellular mechanism whose importance is underlined by its high degree of conservation since this mechanism is conserved throughout the plant and animal kingdoms and species, and has been demonstrated not only in plants but also in the worm Caenorhabditis elegans and yeasts, and mammals—humans and mice.

SUMMARY

We provide a double-strand oligonucleotide including two complementary oligonucleotide sequences forming a hybrid, each including at one of their 3′ or 5′ ends, one to five unpaired nucleotides forming single-strand ends extending beyond the hybrid, one of the oligonucleotide sequences being substantially complementary to a target sequence belonging to a DNA or RNA molecule to be specifically repressed, the target sequence belonging to a DNA or RNA molecule of a gene coding an angiogenic factor.

We also provide a pharmaceutical composition including as active agent at least one oligonucleotide.

We further provide a method for preventing or treating a disease resulting from expression of VEGF gene including administering the pharmaceutical composition.

We still further provide a method for preventing or treating a disease linked to hypervascularization including administering the pharmaceutical composition.

We further yet provide a method for preventing or treating a disease linked to tumoral angiogenesis including administering the pharmaceutical composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the proteins RARα, PML and the associated fusion protein, PML-RARα.

FIG. 1B represents the results of transfections with an siRNA directed against PML-RARα.

FIG. 2A to FIG. 2E pertain to the inhibition of the expression of VEGF by siRNA directed against this protein and the consequences of this inhibition:

FIG. 2A represents the immunodetection of VEGF in the cJ4 or LNCaP cells transfected by the control siRNA or a siRNA directed against VEGF;

FIG. 2B represents the quantification using ELISA of VEGF in conditioned medium of cj4 cells transfected by the control siRNA or the siRNA VEGF as a function of time after transfection;

FIG. 2C represents the growth curve in nude mice of tumors stemming from the subcutaneous injection of 10⁶ cells of cJ4 that is not transfected, transfected by the control siRNA or the siRNA VEGF;

FIG. 2D represents the appearance of the tumors on day 7 after injection of the cells; and

FIG. 2E represents the immunodetection of VEGF in tumors stemming from the injection of cJ4 cells transfected with the control siRNA or the siRNA VEGF after 12 days of development in vivo.

FIG. 3 shows the effect of inhibition by a siRNA specific of the expression of a transcription factor, HIF1α, on the transcriptional response to hypoxia. It also shows the measurement of the activity of a reporter VEGF luciferase in response to hypoxia in CJ4 cells that are not transfected, by the control siRNA or by the siRNA directed against HIF1α.

FIG. 4A to FIG. 4F show the inhibition by siRNA specific of the expression of the androgen receptor in the cells and functional consequences of these inhibitions:

FIG. 4A represents the detection by immunoblot of the expression of the androgen receptor 48 h after transfection of the LNCaP cells by a control siRNA or a siRNA directed against the androgen receptor (AR);

FIG. 4B represents the measurement of the activity of a reporter 4×ARE luciferase to R1881 in various clones of the line LNCaP not transfected, or transfected by control siRNA or by siRNA AR;

FIG. 4C represents the comparison of the response to R1881 of LNCaP cells that were not transfected (100%), and LNCaP cells transfected by a control siRNA, a siRNA directed against the androgen receptor (AR) or a siRNA recognizing specifically a punctiform mutation present in the androgen receptor of the line LNCaP;

FIG. 4D represents the growth in nude mice of tumors resulting from the subcutaneous injection of LNCaP cells transfected by a control siRNA or by a siRNA directed against the androgen receptor;

FIG. 4E represents the growth of LNCaP tumors in mice having received on the 40^(th) day after implantation of the cells an intravenous injection in the tail vein of 2 μg of siRNA directed against VEGF or of control siRNA; and

FIG. 4F represents the growth of LNCaP tumors in mice having received on the 34^(th) and 40^(th) days after implantation of the tumor cells an intraperitoneal injection of 2 μg of siRNA directed against the androgen receptor or control siRNA.

FIG. 5A to FIG. 5K show the inhibition of the expression of wild or mutant forms of p53 by siRNAs and the functional consequences of these inhibitions:

FIG. 5A represents a nucleic acid sequence encoding the human p53 protein (SEQ ID NO: 1);

FIG. 5B represents the specific, dose-dependent inhibition by the siRNAs of the expression of wild or mutant forms of p53 transfected in cells not initially expressing it;

FIG. 5C represents the specific inhibition by siRNAs of the simultaneous or not simultaneous expression of wild or mutant forms of p53 transfected in cells not initially expressing it;

FIG. 5D represents the inhibition of the expression of wild endogenous p53 or a mutant form of 53 transfected by siRNA;

FIG. 5E represents the effect of the inhibition of p53 by siRNAs on the resistance to genotoxic stress;

FIGS. 5 F, G, H and I represent the inhibition of the expression of a mutant form of p53 in the cells of a patient with Li-Fraumeni cancer syndrome at the level of the mRNA (5G) and the expression of the protein by immunoblot (GF) or in indirect immunofluorescence (5H) and the consequences on the resistance of these cells to genotoxic stress;

FIG. 5J shows the inhibition by the siRNAs specific of the dependent transfection of the wild or mutant forms of p53; and

FIG. 5K shows the inhibition of the expression of one of the target genes of p53, p21, inhibitory protein of cellular proliferation, by the coexpression of mutant forms of p53 and the restoration of this expression by treatment of the cells with a siRNA inhibiting the synthesis of the mutant form of p53.

FIG. 6A to FIG. 6D show the inhibition of the expression of the protein E6 of the human papilloma virus HPV by specific siRNAs and the consequences of this inhibition:

FIG. 6A represents the nucleic acid sequence of papilloma virus type 16 (HPV 16) genome and the nucleic acid sequences contained therein encoding HPV proteins;

FIG. 6B represents the effect of inhibition by siRNAs specific of the expression of protein E6 of HPV in cells that express this virus, on the expression of p53 and p21; and

FIGS. 6C and 6D represent the effect of the inhibition of the expression of the protein E6 of HPV on the cell cycle.

FIG. 7A to FIG. 7E the use of hybrid siRNAs comprising DNA bases and RNA bases:

FIGS. 7A and 7B represent the effect of siRNAs DNA/RNA hybrids on the expression of the GFP expression by transfection of the cells;

FIG. 7C compares the effect of RNA/RNA, DNA/RNA and RNA/DNA siRNAs at constant dose on the inhibition of the transcription induced by the androgen receptor; and

FIGS. 7D and 7E represent the effects of a substitution of RNA bases by DNA bases in the siRNA sequence inhibiting the synthesis of p53.

FIG. 8 shows the inhibition of luciferase in tumors expressing this enzyme by injection of siRNA via the subcutaneous, intratumoral, intraperitoneal or intravenous route.

DETAILED DESCRIPTION

We demonstrate that the approach described in more detail herein is much more effective for specifically repressing genes as compared to the techniques employed in the prior art. Moreover, this approach can combine the advantages of antisense and the antigene properties. In one aspect, we have found that in plants cosuppression was effected at the post-transcriptional level on mature RNA and also at the transcriptional level, thus on the gene itself. In another aspect, the repression is transmitted from generation to generation and thus enables repression of a gene in a prolonged definitive manner.

Thus, the disclosure has as one aspect a double-strand oligonucleotide to be used in an interference RNA (RNAi) process, that is comprised of two complementary oligonucleotide sequences each comprising at one of their 3′ or 5′ ends, one to five unpaired nucleotides forming single-strand ends extending beyond the hybrid, one of said oligonucleotide sequences being substantially complementary to a target sequence belonging to a target DNA or RNA molecule which it is desired to repress specifically. This DNA or RNA can be of any type, it can be, e.g., messenger or ribosomal RNA or in one aspect the DNA of a gene.

Each of the two complementary oligonucleotide sequences advantageously comprises the same 3′ or 5′ end with five unpaired nucleotides forming single-strand ends extending beyond the hybrid.

In one embodiment, the two oligonucleotide sequences are advantageously substantially the same size.

Because of the base-pairing law, we designate distinctions by oligonucleotide wherein one or the other of the double-strand oligonucleotide sequences that is complementary to a target sequence belonging to a DNA or RNA molecule that is specifically repressed, can be either a single or double strand.

The oligonucleotides can be of a ribonucleotide, deoxyribonucleotide or mixed nature. In a preferred embodiment the complementary oligonucleotide of the target sequence, also designated antisense strain, is comprised primarily of a ribonucleotide. The sense strain can be of a ribonucleotide, deoxyribonucleotide or mixed nature. Examples of oligonucleotides of the invention of type RNA/RNA or DNA/RNA are given in the experimental part below.

In one preferred embodiment, the RNA/RNA hybrids are more stable than the DNA/DNA or DNA/RNA hybrids and much more stable than the single-strand nucleic acids used in the antisense strategies.

The term oligonucleotide is also understood to mean a polynucleotide of 2 to 100, more generally of 5 to 50, nucleotides of the ribonucleotide, deoxyribonucleotide or mixed type.

The part of the oligonucleotide sequence that is hybridized and complementary to the target sequence preferably is of a size comprised between 15 and 25 nucleotides, most preferably from 20 to 23 nucleotides.

The double-strand oligonucleotides comprise, preferably at the 3′ end of each strand, from 1 to 5 nucleotides, preferably from 2 to 3 nucleotides, and most preferably 2 nucleotides extending beyond the hybrid. These nucleotides extending beyond the hybrid can be complementary to or not complementary to the target sequence. Thus, in a particular form of implementation of the invention, the nucleotides extending beyond the hybrid are any nucleotides, e.g., thymines.

It is possible to represent a double-strand oligonucleotide in the following manner, wherein each hyphen corresponds to a nucleotide and wherein each strand comprises at its 3′ end two thymines extending beyond the hybrid:

-   -   5′ - - - TT3′     -   3′TT - - - 5′.

The sequence of the oligonucleotides is substantially complementary to a target sequence belonging to a DNA or messenger RNA molecule of a gene which it is desired to repress specifically. Although preference is given to oligonucleotides perfectly complementary to the target sequence, the term “substantially complementary” is understood to refer to the fact that the oligonucleotide sequences can comprise several nucleotides mutated in relation to the target sequence as long as the repressive properties of the targeted gene are not changed. Thus, an oligonucleotide sequence can comprise from 1 to 3 mutated nucleotides.

These mutated nucleotides can thus be those extending beyond the hybrid or nucleotides within the oligonucleotide sequence.

In one aspect, an oligonucleotide can be a perfect hybrid or contain one or more mismatches within the double strand. Nevertheless, it is preferable that part of the oligonucleotide sequence, which is hybridized, be perfectly complementary to the target sequence whereas the nucleotides extending beyond the hybrid can be of any type and especially thymines.

The term “perfectly complementary” is understood to mean that the oligonucleotide is complementary to a sequence that belongs to a DNA or RNA of a mutated gene. The oligonucleotides can thereby enable discrimination between the sequence of the wild gene and the mutated gene present a particular value both in the analysis of the genes as well as in the therapeutic uses of the oligonucleotides.

The oligonucleotides are generally comprised of natural nucleotide bases (A, T, G, C, U) but they can also be comprised of modified nucleotides or nucleotides bearing reactive groups or bridging agents or intercalating agents that can react with the target sequence complementary to the oligonucleotide.

The oligonucleotides can be prepared by the conventional methods for the chemical or biological synthesis of oligonucleotides.

We also provide oligonucleotides coupled to substances promoting or enabling their penetration, targeting, or addressing into cells. These substances can for example include lipids, proteins, polypeptides, peptides or any other natural or synthetic substance. In fact, the oligonucleotides are intended to be internalized in the cells and, advantageously in certain cases, into the nucleus of cells where they interact with nucleic acid molecules of nucleic acids bearing the oligonucleotide target sequence. Similarly, it can be of value to promote their penetration into a particular tissue such as a tumor, bone, etc.

The oligonucleotides are useful for repressing in an effective and specific manner a gene or a set of genes, thus allowing for the treatment of numerous human pathologies. They can also be used as a research tool for the investigation and the comprehension of the gene function. We thus achieve pharmaceutical compositions comprising an oligonucleotide or a set of different nucleotides and the use of these oligonucleotides, alone or coupled to transport substances, such as a drug.

The oligonucleotides can be employed in ex vivo applications, e.g., during grafting. Thus, the oligonucleotides can be transfected into the cells, especially tumor cells, which will then be injected or they can be injected into tissues. For example, the oligonucleotides can be injected into already developed tumors via the local, systemic or aerosol route, etc. with vectorization agents.

The oligonucleotides will be used at adequate concentrations in relation to the application and the form of administration employed with suitable pharmaceutical excipients. Depending on the nature of the oligonucleotides (DNA/RNA or RNA/RNA), different doses could be used in order to obtain the desired biological effect.

The oligonucleotides are also useful as diagnostic tools making it possible to establish in vitro the genetic profile of a patient on the basis of a cell sample from the patient. The implementation of the oligonucleotides in such an analysis method makes it possible to know or to anticipate the response of the cancerous cells of this patient and to establish a personalized treatment or to adjust the treatment of a patient.

The oligonucleotides present multiple advantages compared to the conventional chemotherapeutic agents:

-   -   The RNA-RNA hybrids are more stable than the DNA-DNA or DNA-RNA         hybrids and much more stable than the single-strand nucleic         acids used in the antisense strategies.     -   Since they constitute natural compounds, there is no fear of any         immunological reactions or drug-related intolerance.     -   The transfection experiments performed in the framework of the         invention show a better penetration of the RNAi into the tumor         cells than that obtained with plasmids. This point is essential         in the case of tumor cells which are generally very difficult to         transfect.     -   The experiments involving systemic injection of siRNAs in vivo         show a very good penetration of these molecules into the         tissues.     -   It is easy to mix multiple RNAi with each other in order to take         as targets multiple cellular genes at the same time.

In one embodiment, the oligonucleotides and the compositions that contain them are useful for the treatment or prevention of infectious or viral diseases, such as AIDS, and the nonconventional infectious diseases, BSE and Creutzfeldt-Jakob disease. In one preferred embodiment, the oligonucleotides are suitable for treating the viral diseases at the origin of cancers. The table below presents examples of viruses implicated in cancerous pathologies in humans.

TABLE 1 Virus Type of associated human cancer Hepatitis B virus (HBV) Carcinoma of the liver Epstein-Barr virus (EBV) Burkitt's lymphoma, nasopharyngeal cancer, Hodgkins' disease, non-Hodgkins lymphoma, gastric cancer, breast cancer Human herpes virus 8 or Kaposi's sarcoma (KS), primary effusion HHV-8/KSHV lymphoma (PEL), multicentric Castleman's disease (MCD) HPV Neck of the uterus, head, neck, skin, nasopharynx Lymphocyte T virus Type T leukemia (HTLV) Hepatitis C virus (HCV) Carcinoma of the liver

The oligonucleotides and the compositions containing them are also useful for the treatment or prevention of diseases linked to hypervascularization such as age-linked macular degeneration, tumoral angiogenesis, diabetic retinopathies, psoriasis and rheumatoid arthritis.

The research studies performed showed that these oligonucleotides are suitable for repressing harmful genes implicated in canceration and thus most particularly useful for the treatment or prevention of cancers and oncologic diseases in general.

An ideal anticancer treatment should lead to the death of the tumor cell while avoiding resistance phenomena. Cell death can be obtained by:

-   -   Inhibition of cellular division, blocking the cell cycle,     -   Induction of the apoptosis of the tumor cells,     -   Induction of senescence,     -   Induction of necrosis,     -   Induction of differentiation. In this case, the treatment causes         the cell to return to a non-cancerous state.

Thus, we provide an oligonucleotide or a set of different oligonucleotides each containing an oligonucleotide sequence complementary to a target sequence belonging to a molecule of DNA or messenger DNA of a gene whose repression induces apoptosis, or senescence or necrosis or the differentiation of the tumor cells or prevents their division or more than one of these phenomena.

Induction of apoptosis of tumor cells is based on the fact that the function of numerous cellular genes (e.g., members of the family BCL2, BCL XL) is to protect the cells from apoptosis. The loss of expression of these genes induced by RNAi enables passage into apoptosis.

Cell death can also be induced by the loss of adhesion of the cells to the matrix (anoikis). This effect can be obtained by disturbing the balance between proteases and protease inhibitors in the tumors and their stromal environment. This disturbance can also diminish the capacities of tumor cells to invade healthy tissues and metastasize. The siRNAs can thus be used to prevent the synthesis of proteins of the families of the matrix metalloproteases (MMP), membranous matrix metalloproteases, their inhibitors (TIMPs) as well as activators of the protease inhibitors such as, PAI-1, and the proteases themselves such as, urokinase.

Induction of senescence is based on the fact that normal cells can only divide a limited number of times. This number is programmed, for example circa 50 divisions for embryonic fibroblasts. Senesence can also be measured by the length of the telomeres, which get shorter as the cellular divisions advance. Below a certain size, the telomeres are no longer functional and the cell, incapable of division, enters into senescence. However, in germinal cells, this length is maintained constant by the action of an enzyme, telomerase. Telomerase is re-expressed in numerous cancers which enables the tumor cells to multiply indefinitely. A RNAi blocking the expression of telomerase would be without consequence on normal somatic cells and would lead tumor cells into senescence.

Blocking cell division also leads cells to senescence. Blockage can be obtained by inhibiting the essential cellular receptors. Depending on the nature of the cell, these receptors can belong to the class of receptors known as the growth factors (notably, EGF, SST2, PDGF, FGF), whether or not they are mutated, or to the nuclear receptors of hormones (notably, androgens, estrogens, glucocorticoids).

The hormone receptors are frequently mutated in cancers and pertains, in this case, to the use of oligonucleotides recognizing the mutated forms of these receptors, which do not inhibit the synthesis of the wild forms. This makes it possible, for example, in the case of prostate carcinomas that have become resistant by mutation of the androgen receptor, to treat patients via the systemic route with siRNAs that block the synthesis of the mutated receptor without inducing the castration effects linked to the inhibition of the wild forms of the receptor in other organs. In fact, the Applicants' present an example using oligonucleotides recognizing mutated forms of the receptor.

The cell cycle can also be stopped by inhibiting the synthesis of proteins indispensable for its unfolding such as, for example, cyclins, cyclin-dependent kinases, DNA-replication enzymes, transcription factors such as E2F.

Induction of necrosis results from the requirement of the tumor cells for oxygen and nutriments. A tumor initially provides for its development from the preexisting vessels of the host. Beyond 1 to 2 mm in diameter, the cells located at the center of the tumor are in hypoxia. This hypoxia, via the intermediary of a proline hydroxylase, leads to the stabilization of the transcription factor Hif1α, whose sequence, SEQ ID NO. 59, is presented in the attachment, which by attaching itself on the HRE sequences in the promoters of its target genes triggers the hypoxic reaction. This reaction leads to the activation of about a hundred genes enabling activation, notably of the pathway of anaerobic glycolysis, which is the stress response, and angiogenesis. This latter mechanism activates the VEGF gene, whose sequence, SEQ ID NO. 60, is presented in the attachment, which is the principal tumoral angiogenic factor.

The oligonucleotides block, for example, the expression of the transcription factor Hif1α or, for example, that of VEGF making the tumor cells incapable of mounting a hypoxic or angiogenic response. Angiogenesis is a mechanism that is normally repressed in the adult with the exception of the menstrual cycle (uterus ovaries). The inhibition of this mechanism therefore has few consequences for normal tissues.

We also provide an oligonucleotide of which one of the oligonucleotide sequences is substantially complementary to a target sequence belonging to a molecule of DNA or messenger RNA of the gene coding:

-   -   the transcription factor Hif1α;     -   one or more isoforms of VEGF A or a member of the family of this         growth factor.

In certain cancers, the tumoral phenotype results from or is maintained by the expression of a protein normally absent from normal cells. This protein can result from the present or prior expression of a viral genome in the cell such as that of the papilloma virus (HPV) or the hepatitis B virus. This protein can also result from the mutation (punctiform, deletion, insertion) of a normal cellular gene. In this case, it is frequent that the mutated protein thereby produced possesses negative transdominant properties in relation to the normal protein. The specificity of the siRNA enables inhibition of the synthesis of the mutant protein without blocking the synthesis of the wild proteins. Two examples relating to the mutated form of the protein p53 and the androgen receptor are reported in the experimental section below.

The research studies performed demonstrate that these oligonucleotides are particularly suitable in one embodiment for repressing harmful genes implicated in canceration and more particularly those genes leading to the formation of fusion proteins in cancerous cells, such as the fusion protein PML-RAR alpha.

Thus in one embodiment, we provide an oligonucleotides whose sequence is complementary to a target sequence belonging to a gene resulting from a chromosomal translocation so as to inhibit the effects of the fusion protein expressed by this gene. Thus, the target sequence corresponds to the sequence of the junction of the fusion protein.

Table 2 is a nonexhaustive list of the fusion proteins representing therapeutic or diagnostic targets for the oligonucleotides.

TABLE 2 Disease Fusion protein Chromosomal translocatian References APL (acute PML-RARalpha t(15; 17)(q22; q21) De The et al. Cell 1991, 66: 675 promyelocytic PLZF-RARalpha t(11; 17)(q23; q21) Chen Z et al. EMBO J 1993, 12: 1161 leukaemia) NPM-RARalpha t(5; 17)(q32; q12) Redner RL et al. Blood 1996, 87: 882 NuMA-RARalpha t(5; 17)(q13; q21) Wells RA et al. Leukemia 1996, 10: 735 STAT5beta/RARalpha Arnould C et al. Hum. Mol. Genet. 1999, 8: 1741 ALL (acute TEL-AML1 t(12; 21)(p13; q22) lymphoblastic BCR/ABL t(9; 22)(q34; q11) leukaemia) MLL/AF4 t(4; 11)(q21; q23) Domer PH et al. Proc Natl Acad Sci USA 1993, 90: 7884-8 ALL-translocation t(12; 21)(q12; q22) CALM/AF10 t(10; 11)(p12-p13; q14-q21) Dreyling MH et al. Proc Natl Acad Sci USA 1996, 93: 4804 ALL1/AF4 t(4; 11) Janssen JW et al. Blood 1994, 84: 3835 E2A/HLF t(17; 19)(q22; p13) Hunger SP et al. Genes Dev 1992, 6: 1608 AML (acute myeloid TLS/FUS-ERG t(16; 21)(p11; q22) AML(M7) Ichikawa H et al. Cancer Res 1994, 54: 2865 leukemia) MLL-AF10 t(10; 11)(p12-p13; q23) Borkhardt A et al. Leukemia 1995, 9: 1796 MLL-AB11 t(10; 11) Shibuya et al. Genes Chromosomes Cancer 2001, 32: 1 HLXB9-ETV6 t(7; 12)(q36; p13) Beverloo et al. Cancer Res 2001, 61: 5374 MLL-ELL t(11; 19)(q23; p13.1) Rubnitz JE et al. Blood 1996, 87: 4804 CBFbeta/MYH11 inv[16] Tobal K et al. Br J Haematol 1995, 91: 104 AML1-MTG8 t(8; 21) Miyoshi et al. EMBO J 1993, 12: 2715 TEL-TRKC t(12; 15)(p13; q25) Eguch et al. Blood, 1999, 93: 1355 AML1/ETO t(8; 21) Kusec R et al. Leukemia, 1994, 8: 735 CALM/AF10 t(10; 11)(p12-p13; q14-q21) Dreyling MH et al. Proc Natl Acad Sci USA 1996, 93: 4804 ETV6-BTL t(4; 12)(q11-q12; p13) Cools et al. Blood 1999, 94: 1820 CBFbeta-SMMHC inv(16)(p13; q22) Wijmenga C et al. Proc Natl Acad Sci USA 1996, 93: 1630 FUS/ERG t(16; 21)(p11; q22) Panagopoulos I et al. Genes Chromosomes Cancer, 1994, 11: 256 DEK/CAN t(6; 9)(p23; q34) on Lindern M et al. Mol Cell Biol, 1992, 12: 1687 MLL-AF9 t(9; 11)(p22; q23) Super HJ et al. Blood, 1995, 85: 855 MLL-ENL (11q23) Schreiner SA et al. Leukemia 1999, 13: 1525 MLL-AF4 t(4; 11)(q21; q23) Domer PH et al. Proc Natl Acad Sci USA 1993, 90: 7884 MLL-AF6 t(6; 11)(q27; 23) Tanabe S et al. Genes Chromosomes Cancer 1996, 15: 206 MLL-AF17 t(11; 17)(q23; q21) Prasad R et al. Proc Natl Acad Sci USA 1994, 91: 8107 MLL-AFX t(X; 11)(q13; q23) Borkhardt A et al. Oncogene 1997, 14: 195 MLL-AF1p So CW et al. Leukemia 2000, 14: 594 MLL-AF1q t(1; 11)(q21; q23) Busson-Le Coniat M et al. Leukemia 1999, 13: 302 MLL self So CW et al. Cancer Res 1997, 57: 117 MLL-CBP t(11; 16)(q23; p13) Taki T et al. Blood 1997, 89: 3945 AML1-ETO t(8; 21) Erickson P et al. Blood 1992, 80: 1825 MDS/AML NPM-MLF1 t(3; 5)(q25.1; q34) Yoneda-Kato N et al. Oncogene 1996, 12: 265 (myelodysplasia/acute myeloid leukemia) CML (chronic Bcr-Abl/p210 Ben-Neriah Y et al. Science 1986, 233: 212 myelogenous AML1-MDS1-EVI1 (AME) t(3; 21)(q26; q22) Fears S et al. Proc Natl Acad Sci USA 1996, 93: 1642 leukemia) BpALL (cell acute TEL-AML1 t(12; 21)(p13; q22) Golub TR et al. Proc Natl Acad Sci USA 1995, 92: 4917 lymphoblastic leukemia) MPD TEL-JAK2 t(9; 12)(p24; q13) Lacronique et al. Science 1997, 278: 1309 (myeloproliferative TEL-PDGFbetaR t(5; 12)(q33; p13) Jousset C et al. EMBO J, 1997, 16: 69 disease) TEL-TRKC t(12; 15)(p13; q25) Eguch et al. Blood, 1999, 93: 1355 CMML (chronic involving PDGFbetaR t(5; 17)(q33; p13) Magnusson et al. Blood 2001 98: 2518 myelomonocytic HIP1/PDGFbetaR t(5; 7)(q33; q11.2) Ross TS et al. Blood 1998, 91: 4419 leukemia) TEL/PDGFbetaR t(5; 12)(q33; p13) Tomasson MH et al. Blood 1999, 93: 1707 MALT (gastric API2-MALT1 t(11; 18)(q21; q21) Motegi M et al. Am J Pathol 2000, 156: 807 mucosa-associated lymphoid tissue lymphoma) ALCL (anaplastic NPM-ALK t(2; 5)(p23; q35) Waggott W et al. Br J Haematol 1995, 89: 905 large cell SU-DHL-1 t(2; 5) Siminovitch KA et al. Blood 1986, 67: 391 lymphoma) ATIC-ALK inv(2)(p23q35) Colleoni GW et al. Am J Pathol 2000, 156: 781 ALK-related translocation t(2; 17)(p23; q25) Maes et al. Am J Pathol 2001, 158: 2185 MPD NUP98-HOXA9 t(7; 11)(p15; p15) Nakamura T et al. Nat Genet 1996, 12: 154 (myeloproliferative disease) APP (amyloid APP + 1 (38-kDa) Hersberger et al. J Neurochem 2001 76(5): 1308-14 precursor protein) in sporadic Alzheimer's disease (AD) or Down's syndrome primary pleural SYT-SSX1 t(X; 18)(p11.2; q11.2) Crew AJ et al. EMBO J 1995, 14: 2333 monophasic synovial SYT-SSX2 t(X; 18)(p11.2; q11.2) Crew AJ et al. EMBO J 1995, 14: 2333 sarcomas (SS) Dermatofibrosarcoma COL1A1/PDGFB rearrangement t(17; 22) O'Brien KP et al. Genes Chromosomes Cancer 1998, 23: 187 protuberans (DP) ARMS (pediatric EWS-FLII Athale et al. J Pediatr Hematol Oncol 2001, 23: 99 alveolar EWS-ERG t(11; 22)(q24; q12) Sorensen PH et al. Nat Genet 1994, 6: 146 rhabdomyosarcoma) PAX3-FKHR t(2; 13)(q35; q14) Fredericks WJ et al. Mol Cell Biol 1995, 15: 1522 ESFT (Ewing sarcoma PAX7-FKHR t(1; 13)(p36; q14) Barr FG et al. Hum Mol Genet 1996, 5: 15 family of tumors) EWS-WTI t(11: 22)(p13: q12) Benjamin et al. Med Pediatr Oncol 1996 27(5): 434-9 DSRCT (desmoplastic EWS/FI-1 t(11-22)(q24; q12) Fidelia-Lambert et al. Hum Pathol 1999, 30: 78 small round cell tumors) MM IGH-MMSET t(4; 14)(p16.3; q32) Malgeri et al. Cancer Res 2000 60: 4058 (multiple myeloma) MPD (stem cell FGFR1-CEP110 t(8; 9)(p12; q33) Guasch et al. Blood 2000 95: 1788 myeloproliferative disorder) Ewing sarcoma (ES)- EWS-FEV t(2; 22)(q13; q22, t(3; 18) Llombart-Bosch et al. Diagn Mol Pathol 2000, 9: 137 peripheral primitive (p21; q23) neuroectodermal EWS-FLI1 t(11; 22; 14)(q24; q12; q11) Bonin G et al. Cancer Res 1993, 53: 3655 tumor (pPNET) EWS-ERG t(21; 22)(q22; q12) Sorensen PH et al. Nat Genet. 1994, 6: 146 ETV6/CBFA2 t(12; 21)(p12; q22) Fears S et al. Genes Chromosomes Cancer 1996, 17: 127 MLS (myxoid FUS/CHOP t(12; 16)(q13; p11) Rabbitts TH et al. Nat Genet 1993, 4: 175 liposarcomas) EWS/CHOP t(12; 22; 20)(q13; q12; q11) Zinszner H et al. Genes Dev 1994, 8: 2513

Targeting the junction between two genes with an oligonucleotide, for example, the two genes pml and rarα, makes it possible to attain specific inhibition of the fusion protein without affecting the biological role of the natural proteins which can be coded by the second allele. This form of implementation thus encompasses all of the fusion proteins implicated in carcinogenesis, particularly the leukemias. Further, the reciprocal forms as well as all of the variants of the fusion proteins cited in the attachment also constitute targets. In one embodiment, we provide for the use of oligonucleotides, as described above, for the preparation of a pharmaceutical composition intended for the treatment of diseases resulting from the expression of a fusion protein, and in particular for the treatment of cancers.

The present anticancer therapies target the cancerous cells, by different approaches that are employed in isolation or combined with each other (chemotherapy, surgery, radiotherapy, immunotherapy). The therapeutic failures are massively due to either the cells not having been reached by the treatment or, primarily, by cells that are mutated in response to the treatment. The capacity for mutation is greatly facilitated by the genetic instability of the tumor cells. The inhibition of tumor vascularization, depriving the cells of oxygen and nutriments, has in the past several years opened new therapeutic perspectives in cancer research. This strategy, which is complementary to the previously mentioned methods, targets the normal endothelial cell of the host, which is genetically stable and theoretically not likely to mutate. Numerous clinical trials directed at inhibiting tumoral angiogenesis via different approaches are underway worldwide. However, the initial reported results appear to be rather disappointing.

We demonstrated that tumors are capable of compensating for the effects of angiogenesis inhibitors by selecting subpopulations of cells secreting strong concentrations of pro angiogenic factors.

Tumors are not comprised of homogeneous cells with regard to their genetic expression. This is attested to by a very large number of studies in which immunolabeling was performed for a large variety of antigens in the tumors. Macroscopically, a tumor is frequently composed of regions that are highly vascularized alongside zones of necrosis or avascular regions.

This tumor heterogeneity promotes the ability of tumors to escape from the applied treatments, no matter what their nature. The greater the diversity of the genetic expression in a tumor, the greater the probability that there exists at least one cell capable of resisting an antitumor agent. It therefore appears to be essential to combine different strategies in order to first reduce the tumoral heterogeneity and avoid the escape phenomena.

We also provide siRNAs that are inhibit the expression of genes responsible for the inactivation of p53 and their use in the treatment of cancers. p53 is the product of a tumor-suppressor gene or anti-oncogene, mutated in more than 50% of the tumors in humans. p53 is thus considered to be a guardian of the genome. It is activated in the cells in the case of genotoxic stress and participates in various processes including the induction of the programmed death process.

In 74% of the cases of monoallelic mutation, the inactivation of p53 is due to a punctiform mutation leading to the expression of protein that is mutated but of normal size. It is generally considered that the mutated version forms heteromers with the product of the wild allele on which it acts as a negative transdominant that blocks its activity. The mutant form also appears to have an oncogenous activity in itself. Thus, the mutated forms of p53 are capable of activating the gene MDR, which facilitates the resistance of the cancerous cells to chemotherapy. Moreover, the expression of mutants of p53 is associated with a stronger tumoral angiogenesis, probably because the mutant forms of p53 are no longer capable of stimulating the transcription of the gene of thrombospondin, one of the most powerful repressors of angiogenesis, and activate VEGF and bFGF, two powerful activators of angiogenesis. Moreover, the cells in which a mutated form of p53 is expressed lose various levels of regulation. In particular, they are no longer capable of initiating a programmed death process which constitutes one of the major protection processes against tumorigenesis. The restoration of wild type p53 activity in cultured tumor cells leads to the restoration of this cellular response. Thus, inhibiting the expression of the mutated forms of p53 represents a potentially powerful tool in anticancer therapy.

At present, there is no effective means for restoring p53 activity in human cancer cells. With regard to the cancers in which both alleles are inactivated, attempts to restore the p53 activity by gene therapy are envisaged. These approaches are complicated by the use of viral vectors that at present do not appear to be very effective.

Furthermore, it has been observed specifically in cervical cancers linked to infection by the HPV virus of the cells of the neck of the uterus that p53 can be inactivated by the overexpression of a viral protein. In fact, this virus codes for a protein, the protein E6, which inactivates p53. In this type of cancer, it is the inhibition of the protein E6 which could restore a wild p53 activity.

We provide new means enabling activation of p53 by inhibiting the expression of the genes responsible for its inactivation. The research studies performed demonstrated that it was possible to repress in a very effective and very specific manner the expression of a mutant form of p53.

We provide oligonucleotides presenting a sequence complementary to a specific polynucleotide sequence of the gene of the mutated p53. Thus, these are oligonucleotides whose sequence bear a mutation in relation to the sequence of wild p53. The sequence of the wild gene of p53 is shown in the attached sequence listings as SEQ ID NO. 1. The different mutations that can intervene in the sequence of p53 are indicated in Table 3.

TABLE 3 Codon Event Codon Event Codon Event Codon Event Codon Event 248 G->A 129 C->A 189 C->G 217 Stop at 219 202 Ins 248 C->T 281 A->G 290 G->T 239 Stop at 259 247 Ins 282 C->T 293 Fr. 136 Stop at 169 187 G->C 171 Ins 175 G->A 157 DEL 201 Stop at 208 273 Stop at 343 203 Ins 196 C->T 161 C->A 275 Stop at 344 182 C->T 290 Stop at 303 213 G->A 195 A->T 132 Stop at 148 263 Stop at 344 233 del 234 T->C 197 G->C 176 Stop at 180 307 Stop at 344 210 Stop at 244 237 T->G 342 Fr. 191 del 261 Stop at 344 201 G->A 244 G->T 135 G->C 218 G->A 285 Stop at 344 92 Ins 256 A->G 145 T->A 234 T->A 159 G->A 44 Fr. 259 A->G 276 G->C 136 C->A 168 C->T 109 ins 260 T->G 173 G->T 245 G->C/G->A 230 C->T 279 G->A/G->A 245 G->T 270 T->G 126 Stop at 148 228 A->C 168 Stop at 170 278 C->T 158 G->C 259 G->C 230 C->A 153 Stop at 178 134 T->A 152 Fr. 171 G->C 287 Stop at 300 247 C->A 194 C->T 132 G->T 197 T->A 269 Stop at 343 272 Stop at 305 273 G->A 288 A->C 236 T->G 227 Stop at 227 137 Stop at 169 309 C->T 247 A->T 239 C->A 231 Stop at 238 148 Stop at 180 274 T->A 273 G->C 288 A->T 275 G->C 157 Stop at 180 156 G->C 283 G->C 161 Fr. 142 T->C 191 Stop at 208 245 G->A 109 Fr. 164 Fr. 312 C->G 243 Stop at 260 193 A->G 174 G->C 142 Stop at 148 282 C->G/G->C 251 C->A 229 T->A 300 C->G 240 A->C 235 Stop at 244 242 C->A 237 G->A 205 A->G 137 T->C 156 Stop at 179 244 C->A 277 G->T 224 G->T 100 G->A 207 A->G 239 C->G 194 T->G 168 A->T 106 C->G 179 Stop at 246 142 T->A 242 G->C 167 Fr. 215 A->G 210 Stop at 214 248 C->A 246 G->C 136 C->G 246 Fr. 315 T->C 177 Stop at 246 68 G->T 164 A->C 117 G->A 229 Stop at 229 296 Fr. 147 T->A 179 C->G 271 Stop at 344 167 A->C 303 C->T 151 C->A 187 G->T 324 T->G 256 Stop at 343 140 C->A 209 A->T 201 Stop at 246 346 Fr. 176 Stop at 176 268 C->T 213 C->T 213 C->A 174 Stop at 246 309 Stop at 336 254 A->C 214 A->G 238 G->T 170 Stop at 177 270 Stop at 344 291 G->A 248 G->T 113 T->G/C->T 234 T->G 129 G->A 139 Stop at 148 266 G->T 143 G->T 354 A->G 46 Stop at 50 251 A->G 273 C->T 160 G->T 259 Stop at 344 160 T->G 221 Stop at 224 273 G->T 198 G->T 319 Stop at 344 56 A->T 237 A->T/G->T 282 C->G 203 G->T 332 Ins 74 Stop at 144 234 Stop at 234 334 G->T 238 T->A 340 Ins 118 A->G 215 T->A 342 C->T 272 G->C 177 del 257 del 191 T->A 132 A->C 276 Ins 179 T->A 192 G->T 290 C->A 249 G->C 277 T->G 190 Stop at 246 294 G->T/G->C 60 A->T 280 G->A 302 G->T 254 Ins 240 del 93 C->A 285 G->A 131 C->G 194 C->A 306 G->T 143 T->C/G->C 241 C->T 168 A->G 172 T->A 175 C->G/G->A 319 G->T 249 G->T 258 G->T 173 G->T/T->G 246 Stop at 261 110 Stop at 122 158 G->A 278 C->A 261 T->C 279 Stop at 305 190 T->G 163 T->C 285 G->C 266 A->G 146 T->G/G->T 192 C->G 176 G->A 287 G->A 199 Fr. 154 Stop at 169 126 T->G/A->G 206 Fr. 294 Fr. 236 C->G 132 del 273 Stop at 305 234 A->G 236 Fr. 168 C->G 175 Stop at 175 266 Stop at 344 238 G->A 301 Ins 201 G->C 152 Stop at 165 64 Stop at 122 254 A->G/T->A 228 A->G 203 Stop at 208 260 Stop at 262 103 C->A 287 G->T 175 Fr. 250 C->A/C->G 194 Stop at 246 343 A->G 143 Fr. 282 G->T 283 G->T 170 C->A 317 Stop at 344 205 A->T 152 Stop at 180 256 C->A 213 C->G 125 Stop at 148 262 Fr. 177 C->G 245 C->T 213 A->C 239 A->G/C->T 171 G->T 216 T->A 342 G->C 232 A->G 119 Fr. 126 C->G 232 T->G 243 G->A 294 G->C 162 T->G/C->G 138 Fr. 275 Stop at 305 296 A->G 240 T->A 12 C->A/C->G 223 C->T 216 G->T 68 G->C 223 C->G 247 C->G 274 G->T 137 Fr. 102 C->T 171 A->C 190 T->A 218 Fr. 251 T->G 104 G->C 328 T->G 240 T->C 246 A->G 252 Ins 117 G->C 150 C->T 315 C->T 250 Fr. 254 T->A 175 Stop at 246 252 C->A 313 C->T 143 T->C 49 G->C 138 Stop at 169 256 Stop at 342 42 G->T 173 G->A 53 G->T 215 T->G 200 Stop at 246 73 G->T 242 G->T 60 C->T 247 Stop at 262 239 del 231 C->A 190 Fr. 202 G->T 104 C->T 215 A->T 172 Stop at 173 246 T->C 204 A->G 297 A->C 147 Stop at 169 211 Stop at 214 157 G->T 265 T->A 252 T->C 276 G->A 150 Stop at 180 239 Fr. 135 T->C 276 C->T 210 A->C 145 C->A 240 A->T 147 G->A 349 A->C 182 T->C 335 C->G 238 T->C 153 C->T 173 G->A/T-> 161 G->A/C->T 285 G->A/A->G 35 Stop at 42 170 G->T G/G->T 83 C->A 85 Stop at 122 47 C->T 260 C->T 225 G->C 304 Stop at 344 98 C->T 89 Fr. 255 Stop at 263 250 del 225 ins 113 C->T 102 Fr. 139 G->C 224 A->G 314 Stop at 344 87 Stop at 148 141 C->G 234 A->C 166 T->A 301 A->G 97 C->T 144 C->T 152 C->A 156 C->A 224 G->A 217 Stop at 246 146 G->A 170 C->T 291 A->G 112 C->G 226 G->T 158 G->T 175 G->C 305 A->G 163 C->A 278 T->A 161 G->A 240 A->G 306 A->T 299 T->A 145 C->T 164 G->T 259 G->T 296 C->T 251 del 133 Stop at 148 165 Ins 87 Fr. 267 del 162 Stop at 180 136 A->C 176 C->G 142 Fr. 151 Stop at 169 44 G->T 239 A->T/C->A 191 Fr. 175 C->G 228 Stop at 238 177 C->A/C->T 245 G->A/C->A 215 G->T 126 A->G 165 Stop at 180 236 A->C 252 T->A 217 G->T 128 T->G 176 C->A 243 T->C 244 G->A/C->A 220 A->G 128 C->T 192 A->G 137 G->A 299 Stop at 305 224 G->C 134 T->C 167 Ins 218 G->C 305 A->T/G->A 242 C->G 172 Fr. 166 T->C 277 G->C 310 A->C 259 A->T 237 T->A 120 A->G 54 Fr. 322 C->G 267 G->C 193 A->C 150 C->A 40 Ins 323 Stop at 344 291 A->T 213 Fr. 155 A->C 156 Stop at 166 315 C->G 298 G->T 246 G->A 203 Stop at 246 168 C->G/A->T 308 G->C 182 C->A 235 Fr. 221 A->G 249 G->T/G->T 323 T->G 233 Stop at 329 Fr. 50 Stop at 109 158 C->A 201 T->A 239 155 A->G 191 Stop at 243 209 G->T 190 T->C 173 T->C 7 G->C 205 T->C 184 Stop at 207 278 T->C 251 A->C 56 G->T 210 Stop at 246 146 G->T 305 G->C 219 Fr. 104 Fr. 110 C->G 250 C->A 176 del 280 A->T 245 G->A/G->A 166 C->A 74 Stop at 122 217 T->G 126 T->A 317 C->T 269 G->T 225 del 174 A->C 132 G->C 125 G->A 155 Stop at 179 253 C->A 289 C->G 181 C->T 214 Fr. 155 Stop at 169 269 INS 234 C->G 184 G->T 248 G->C 156 Stop at 169 184 T->C 232 A->C 220 T->C 307 Ins 162 C->T 304 T->G 317 A->T 266 G->A 152 G->T 196 A->G 204 A->C 132 Fr. 279 G->A 178 C->G 213 Stop at 246 66 Stop at 145 299 Fr. 305 Ins 253 C->T 214 C->T 259 Stop at 263 158 C->G/G->T 220 A->C 270 T->C 269 C->T 263 Stop at 271 142 Stop at 169 284 A->C 281 C->A 287 A->G 280 Stop at 344 203 Stop at 207 280 G->C 216 Fr. 313 C->G 237 Stop at 246 248 G->C/G->C 172 Stop at 131 Fr. 108 Stop at 144 289 C->A 256 A->C 231 141 Ins 321 A->G 315 Stop at 344 262 Stop at 343 174 Stop at 140 Fr. 244 C->T 312 C->T 301 Stop at 343 176 163 T->A 198 Stop at 246 145 C->G 335 G->A 224 Ins 178 A->C 135 Ins 169 G->T 179 Fr. 251 Stop at 186 G->T 187 Stop at 246 184 G->A 341 Stop at 344 344 208 A->T 264 del 364 G->A 103 C->G 261 del 255 Fr. 52 C->T 144 del 159 Stop at 179 181 G->A 307 G->A 141 G->C 146 Stop at 169 189 Stop at 246 265 C->T 130 T->G 167 A->G 190 C->A 274 Stop at 304 272 T->C 356 G->T 84 C->T 249 A->C 149 Fr. 136 C->T 43 T->C 122 Stop at 169 214 T->A 183 Stop at 183 281 G->T 159 G->C 140 A->T 204 G->A 227 Stop at 245 316 C->T 280 Ins 153 Ins 242 G->A/C->G 292 Stop at 343 130 C->G 327 Fr. 173 Fr. 208 Stop at 241 178 A->G 234 C->A 87 C->A 186 Fr. 158 Stop at 180 251 Stop at 343 368 Fr. 156 G->T 152 C->G 217 Stop at 221 252 Stop at 263 301 Fr. 158 C->G 171 A->G 262 Stop at 344 64 Fr. 148 Fr. 161 G->T 180 G->T 239 Stop at 246 89 Stop at 122 176 G->T 173 Stop at 180 202 G->A 205 Stop at 246 108 Stop at 122 152 C->T 199 G->T 227 T->G 214 T->C 110 Ins 248 C->G 144 Fr. 298 G->A 297 ins 124 Stop at 124 255 T->G 233 Fr. 303 G->A 268 Fr. 285 del 271 Fr. 275 T->G 261 Ins 256 A->T 342 del 274 Fr. 162 T->G 276 C->G 223 C->A 313 A->T 225 G->A 178 Fr. 305 Fr. 26 Stop at 43 217 T->A 176 T->A 256 Fr. 117 Stop at 122 186 A->T 167 Stop at 169 135 Fr. 225 Fr. 155 Stop at 177 214 Stop at 246 278 C->T/T->C 135 C->G 148 T->A 277 T->A 245 C->A 290 Stop at 304 151 C->T 187 G->A 298 A->C 287 G->C 173 Stop at 173 159 C->T 250 C->A/C->A 141 C->T 96 C->T 259 C->T 179 A->G 254 T->G 115 T->C 164 Stop at 166 288 T->A 306 C->T 257 T->C 119 G->A 255 Ins 207 T->A 174 G->A 275 T->C 120 Fr. 275 del 197 Stop at 208 208 Fr. 216 G->T/G->T 127 T->A 284 ins 214 A->T 126 Fr. 149 T->C 133 Fr. 161 G->C 127 C->G 173 del 240 G->T 144 A->T 246 A->T/G->T 337 G->C 192 C->T 65 A->T 187 T->C 199 G->C 102 Stop at 122 209 Fr. 125 C->T 205 T->A 195 Stop at 246 187 Stop at 202 216 T->G 166 C->T 209 A->G 275 Fr. 100 A->G 258 G->A 242 C->T 237 A->T 283 Stop at 305 140 Stop at 143 282 G->C 263 A->C 337 G->T 233 ins 176 Stop at 179 308 Fr. 139 G->T 342 G->A 127 Stop at 169 235 Ins 332 Fr. 165 A->T 377 C->A 138 G->A 250 Stop at 262 173 T->G 241 T->G 93 C->T 208 A->T/C->T 284 Stop at 305 249 Fr. 255 T->A 202 C->T 106 del 132 G->A 275 G->A 265 Fr. 199 Stop at 246 245 G->C/G->T 129 C->T 294 G->T 279 Fr. 252 C->T 212 Stop at 246 210 C->T 316 Fr. 241 Fr. 254 C->T 133 G->A 232 C->T 159 C->A 151 C->G 262 G->A 124 T->C 257 C->T 118 Ins 156 Fr. 263 A->G 51 Stop at 122 164 Stop at 169 277 G->A 170 A->T 274 Stop at 344 170 G->A 249 del 244 G->C 204 G->T 293 Stop at 344 150 del 187 Fr. 264 Fr. 249 A->G 156 C->G 85 Stop at 143 210 Fr. 278 C->T/C->T 280 G->T 157 Stop at 169 195 T->A 207 T->C 177 C->T 281 A->C 92 C->T 314 Stop at 338 226 G->C 179 C->T 94 T->A 201 G->T 307 Stop at 340 168 C->G/C->G 281 C->T 153 C->A 202 Stop at 246 67 Stop at 122 185 A->G 141 G->A 172 T->C 222 C->T 255 Stop at 344 198 Stop at 208 283 Stop at 173 T->A 223 Stop at 246 163 del 208 G->C 344 296 C->G 264 Stop at 344 191 Stop at 246 331 A->C 136 Stop at 284 A->G 273 C->A/G->A 255 Stop at 257 320 Stop at 336 148 135 G->T 316 C->A 262 Stop at 263 331 A->C/G->A 286 G->A 31 G->A 271 Ins 264 C->A 338 T->A 109 C->A 72 Fr. 129 Fr. 348 G->T 280 Fr. 164 A->G 91 G->A 192 Ins 232 Stop at 246 290 Fr. 238 G->C 110 Fr. 307 G->T 170 ins 297 Fr. 110 G->T 154 Fr. 220 T->A 114 T->A 297 C->G 113 T->G 158 Fr. 285 A->G 343 G->T 136 Stop at 164 162 C->G 167 C->T 226 G->A 26 Stop at 36 149 Stop at 180 183 C->G 178 Stop at 180 137 Ins 137 G->T 221 Stop at 246 287 Stop at 195 Stop at 208 259 Ins 145 G->C 228 ins 344 197 G->A 234 Fr. 146 T->C 243 Stop at 340 152 G->A 199 G->A 135 del 286 A->T 292 Stop at 304 138 C->T 227 Ins 102 Stop at 116 296 A->T 328 del 278 C->G 248 G->T/G->T 324 G->A/A->G 164 G->A 338 Stop at 346 236 T->C 265 T->C/G->T 27 Fr. 148 T->G 243 A->C 237 A->G 272 T->A 162 del 274 del 348 T->A 289 T->A 274 T->G 277 Ins 211 Stop at 215 304 C->T 237 G->T 349 Fr. 135 T->G 239 A->T 228 G->T 136 Ins 203 Fr. 69 C->G 313 ins 370 A->C 99 Stop at 205 T->G 242 T->G 327 T->G 149 C->T/C->T 147 205 A->C 157 G->A 211 C->A 158 C->T/G->A 134 Stop at 246 A->T 198 G->C 246 Stop at 246 240 G->A 169 282 Stop at 305 157 T->G 163 C->T 258 Stop at 263 242 T->C 133 A->C 279 G->C 252 del 317 C->A 193 C->T 162 A->T 134 Ins 129 del 262 T->A 188 Fr. 174 A->T 239 Stop at 263 215 G->C 263 A->T 152 Stop at 253 C->G 168 Stop at 169 253 A->C 163 T->G 169 131 A->G 134 Fr. 274 Ins 312 Fr. 57 Fr. 137 T->A 253 A->T 154 C->A 301 C->A 281 C->G 141 Fr. 254 A->T 183 C->T 226 G->A/G->A 260 Stop at 157 T->A 247 A->G 225 T->A 200 A->C/A->C 263 157 Fr. 235 A->T 149 ins 207 G->C 132 A->T 176 Ins 176 Stop at 243 171 Fr. 226 Stop at 227 249 Stop at 240 Fr. 163 Stop at 169 287 A->T 266 263 274 T->C 248 Stop at 344 133 G->T 113 del 167 G->T 46 Fr. 289 Stop at 304 137 C->A 226 Fr. 17 A->T 112 Fr. 163 Fr. 148 G->T 94 C->A 24 A->T 295 C->T 207 Fr. 246 A->C 127 Fr. 175 C->T 193 T->A 251 A->T 251 T->C 133 Stop at 145 358 G->A 221 G->T 112 Stop at 120 273 T->C 153 Stop at 180 175 Ins 227 Fr. 120 Stop at 122 297 C->A 75 C->T 115 C->T 241 C->A 231 C->T 192 G->A 116 Stop at 122 103 Fr. 281 G->A 212 Stop at 214 244 C->G 184 del 237 Fr. 316 Ins 179 A->C 221 Stop at 222 106 Stop at 122 250 C->T 344 Fr. 174 G->T 243 T->A 69 Stop at 147 365 A->G 145 T->G 232 del 308 C->G 298 Stop at 344 271 G->A 145 T->C 173 Stop at 195 189 C->A 182 ins 320 G->C 194 T->C 273 Stop at 344 239 133 del 349 G->T 162 A->G 143 T->A 142 C->G 163 Stop at 168 126 del 315 Ins 161 C->T 295 C->A 174 del 36 G->A 203 T->A 72 Stop at 120 156 Stop at 168 330 T->G 76 Fr. 273 Ins 265 del 213 Stop at 245 125 C->G 241 C->G 62 G->T 214 Stop at 214 243 Stop at 244 258 Stop at 344 281 G->C 71 Fr. 107 Stop at 147 289 C->T 330 Stop at 335 244 G->A 128 Fr. 317 Ins 211 del 113 T->C 218 T->G 203 T->C 165 C->A 220 Stop at 244 265 T->G 256 Stop at 254 C->G 99 Stop at 122 229 T->G 126 T->C 344 282 Fr. 36 C->T 253 del 214 Stop at 218 280 A->C/G->C 258 G->C 245 Ins 302 Stop at 303 284 C->T 258 A->G 217 Fr. 76 C->T 208 A->G 96 ins 270 T->A 139 A->C 160 T->A 212 Stop at 244 62 Stop at 121 176 T->G 215 A->C 165 A->C 129 Stop at 145 285 A->C 171 Stop at 243 Ins 269 Fr. 190 del 358 G->T 231 295 Fr. 245 G->T/C->A 216 Stop at 221 122 G->A 251 Stop at 285 A->T 208 G->A 275 Stop at 304 69 Stop at 122 263 170 Stop at 179 236 C->T 150 ins 155 C->T/C->G 337 C->T 208 Stop at 246 294 G->A 188 C->G 245 G->A/C->G 266 G->C 209 Stop at 214 251 Fr. 220 Ins 181 C->G 203 G->C 240 Stop at 263 215 Ins 292 A->C 185 Ins 241 Stop at 141 G->T 154 C->T 305 G->T 52 Stop at 56 252 151 Fr. 293 G->C 48 A->T/C->T 112 Stop at 122 193 A->T 182 Stop at 246 161 C->G 154 C->G 165 Stop at 169 255 A->G 140 A->G 56 G->A 150 Fr. 323 del 194 C->G 142 C->T 139 G->A 329 C->A 67 C->G 342 Stop at 169 T->A 222 G->C 80 C->T 148 A->T 342 170 A->G 302 Stop at 344 243 T->G 230 Stop at 246 55 Ins 271 A->G 144 Stop at 169 104 Stop at 148 95 Stop at 148 257 C->G 331 C->T 166 T->G 117 Stop at 148 276 Stop at 286 282 ins 194 Stop at 245 149 T->A 138 C->A 249 Stop at 342 245 G->C 113 T->G/T->G 204 del 248 C->T/G->C 208 del 209 ins 165 C->T 127 C->A 167 G->A 163 Stop at 163 239 ins 176 Stop at 246 289 T->C 214 C->G 213 A->G 179 T->G 207 T->G 261 A->G 272 Fr. 282 Stop at 304 314 C->T 297 C->T 269 A->G 186 A->G 295 Stop at 344 155 C->T 141 T->G 128 Stop at 169 147 Ins 160 del 249 Stop at 181 G->C 159 Stop at 169 261 T->G 233 A->T 344 229 Fr. 204 Ins 240 T->G 186 T->C 116 C->G 276 Fr. 242 Stop at 242 288 Fr. 243 T->C/G->A 163 A->G 149 Stop at 169 237 del 286 A->G/A->T 142 C->A 173 G->C 193 C->G 284 Stop at 304 126 Stop at 169 144 G->T 255 C->T 293 Stop at 304 331 G->T 182 Fr. 231 Fr. 255 C->G 147 Fr. 130 T->A 298 Fr. 254 Fr. 218 T->C 286 G->T 39 C->T 220 T->G 266 ins 301 Stop at 287 Stop at 303 352 G->C 269 G->A 258 A->C 344 293 G->A 209 Stop at 246 232 Fr. 239 Stop at 261 271 A->T 295 T->C 90 C->T 131 del 262 G->C/G->C 286 A->G 215 Fr. 111 G->A 261 Fr. 296 Stop at 334 294 A->G 333 Fr. 119 C->T 111 T->A 284 del 264 C->T 28 A->C 141 T->C 285 Fr. 150 A->C 235 A->G 67 C->T 202 T->C 266 G->A/A->T 225 G->T 249 A->T 288 A->G 326 A->G 162 Stop at 169 247 Fr. 216 G->A 276 del 36 G->T 208 Ins 322 C->T 215 G->A 292 Fr. 68 A->G 250 Ins 85 Stop at 117 272 G->A 189 C->T 117 G->T 130 Stop at 169 86 ins 267 Stop at 210 A->G 145 G->T 289 Fr. 189 Fr. 344 217 T->C 215 G->A/T->A 198 Fr. 315 Fr. 242 G->A 135 Stop at 169 325 G->A 302 Fr. 169 Stop at 180 195 T->C 165 A->G 112 G->A 137 C->T 245 G->T/G->T 172 G->T 234 del 308 G->A 191 Ins 175 C->A/G-> 239 A->G 218 del 63 C->T 186 G->A C/C->G 262 G->T 100 C->T 104 A->T 237 ins 71 C->T 255 T->C 169 G->A 212 T->C 230 A->G 72 Stop at 148 286 A->C 158 Stop at 179 217 G->A 184 A->G 98 T->A 283 G->A 143 Stop at 169 328 T->C 157 ins 287 Stop at 304 190 C->T 200 Ins 45 C->T 95 T->A/T->G 162 A->G/C->T 154 G->A 185 Stop at 246 299 G->C 314 Fr. 130 T->C 272 Stop at 11 G->A 111 T->C 306 A->G 215 Stop at 243 344 217 G->C 127 T->C 45 C->A 204 Stop at 207 143 G->A 72 Stop at 122 162 Ins 100 Fr. 315 Stop at 336 271 A->C 105 G->T 360 G->T 162 Fr. 33 Stop at 43 133 T->A 221 G->A 257 Ins 319 Fr. 41 Stop at 43 174 Fr. 253 A->G 341 T->G 113 Fr. 80 Stop at 120 132 A->G 300 Stop at 344 242 Stop at 246 126 C->A 96 Stop at 147 252 Fr. 250 Stop at 342 262 del 196 Stop at 246 207 Stop at 212 330 T->A 135 T->A 257 T->G 175 G->A/C->G 215 Stop at 245 179 C->A 159 C->T/C->T 229 T->C 182 T->G 224 Stop at 246 309 C->G 249 G->A 196 G->A 190 C->G 260 del 212 ins 198 A->G 200 A->G 141 Stop at 148 276 Stop at 339 175 G->T 238 T->G 278 Stop at 344 166 Stop at 180 290 Stop at 339 153 C->G 243 A->T 144 G->C 345 Stop at 369 300 Stop at 343 145 C->G/G->T 259 G->A 158 Stop at 169 192 C->A 51 Stop at 121 277 Fr. 268 A->G 252 Stop at 344 65 Fr. 301 Stop at 303 275 G->T 287 Fr. 241 Stop at 261 185 G->T 236 A->C/C->G 110 C->T 302 G->A 282 C->T/G->A 181 C->A 83 C->T 232 T->A 189 G->C 276 G->T 190 Stop at 208 237 T->C 151 C->A/C->T 212 Fr. 196 C->A 155 C->G 156 ins 218 G->T 51 G->T 193 T->C 242 G->T/C->T 128 C->G 139 A->G 160 G->C 160 A->C 269 A->T 243 T->G/G->C 250 C->G 207 Ins 243 A->G 283 Fr. 133 A->G 280 A->C 147 T->G 206 Stop at 246 189 G->A 125 C->A 127 C->T 177 Fr. 194 T->A 244 G->A/G->C 62 A->G 176 G->C 121 Fr. 212 T->A 138 Stop at 148 54 C->T 274 G->C 147 T->C 169 A->G 188 Stop at 208 84 C->G 246 T->G 160 A->G 183 T->C 246 del 202 G->C/T->G 229 Stop at 230 Fr. 77 C->G 180 G->C 319 A->C 238 237 A->C 188 Ins 175 del 138 C->G 247 A->C 47 Stop at 121 158 G->T/C->T 290 Stop at 301 229 T->A/G->A 290 G->A 78 Fr. 194 Stop at 207 271 del 101 Stop at 122 219 Stop at 81 Stop at 122 253 Ins 156 Stop at 180 278 Stop at 304 246 108 Stop at 146 360 Stop at 369 69 C->A 339 Fr. 88 Stop at 110 G->C 191 C->G 112 C->A 303 G->T 122 156 C->T 141 T->A 193 C->A 247 Stop at 344 254 T->C 217 del 303 A->T 222 Fr. 299 Ins 283 C->G 242 T->A 49 Ins 228 Stop at 245 293 del 299 G->A 245 Stop at 340 62 Stop at 141 145 del 247 Stop at 343 346 G->A 251 Ins 103 del 148 Stop at 167 5 C->T 116 T->C 91 G->T 105 del 140 Stop at 168 123 C->T 150 A->G 136 A->G 121 del 171 Stop at 180 126 C->T 95 T->C 146 G->C 124 Ins 304 Fr. 320 G->A 54 T->A 164 A->T 124 Stop at 167 159 ins 356 G->A 256 C->T 194 Fr. 338 Stop at 343 261 G->A 379 G->A 309 C->A 255 A->T 336 G->T 304 A->G 154 Stop at 180 109 T->C 339 Ins 124 C->G 222 G->T 164 G->C 265 T->C 35 G->T 284 A->T 291 G->C 75 C->G 139 Stop at 213 G->T 144 G->A 147 T->A/T->A 163 Stop at 165 169 261 Stop at 263 227 C->T 216 G->C 238 Stop at 244 154 G->T 299 T->C 208 G->T 91 Ins 8 C->T 179 A->T 204 A->T 228 G->A 311 A->C 15 A->C 255 del 47 Fr. 196 G->T 334 Stop at 344 61 A->G 342 Stop at 178 C->A 195 C->G 211 T->C 72 C->T 344 257 G->A 272 T->G 197 G->T 102 ins 11 G->C 341 C->T 53 G->C 202 C->A 104 G->T 121 Stop at 290 C->T 290 C->G 219 C->A 106 A->G 122 169 T->C 292 A->T/A->T 228 G->C 365 C->T 34 ins 233 C->T 245 Stop at 246 163 A->C 10 C->T 53 G->A 198 G->A 188 Stop at 246 271 G->C/A->G 21 C->T 144 A->C 200 Fr. 288 Stop at 344 238 Fr. 361 G->A 280 A->G 228 C->G 176 Fr. 206 T->A 364 C->T 326 G->T 236 C->A 148 Stop at 179 52 del 385 T->C 332 Stop at 245 G->T/C->T 161 Stop at 169 94 Stop at 122 307 A->G 344 249 Ins 211 Stop at 246 236 Stop at 236 161 G->T/C->T 256 Ins 251 T->A 244 Stop at 246 107 C->A 241 Stop at 263 283 C->T 258 Fr. 247 del 106 Fr. 327 Stop at 335 232 T->C 278 Ins 260 Stop at 344 69 Fr. 157 T->C 184 Fr. 279 Ins 216 T->C 204 Fr. 132 Stop at 169 273 C->G 296 A->C 231 A->T 305 A->C/G->T 221 A->C 133 T->C 255 Stop at 343 208 C->A 269 Stop at 344 184 G->C 272 G->T 290 Stop at 344 301 C->G 230 Stop at 238 157 Stop at 179 293 G->T 137 Stop at 145 208 C->G 227 Stop at 228 289 Stop at 305 267 G->A 155 Fr. 237 G->C 363 G->A 105 G->C 325 G->T 206 Ins 243 G->C 253 Fr. 215 Stop at 221 71 Ins 242 Ins 159 G->T 250 Stop at 344 179 Stop at 180 120 A->T 300 Fr. 33 Ins 259 C->A 128 Stop at 148 151 Ins 191 T->C 192 del 167 del 256 Stop at 263 307 Fr. 191 C->A 312 C->A 173 Stop at 246 131 Stop at 169 108 Fr. 246 T->A 321 Stop at 344 313 Fr. 143 Stop at 167 257 T->A 258 A->T 283 C->G/C->G 346 Ins 158 C->T/G->T 257 Stop at 143 Ins 285 G->T 293 Ins 207 A->T 344 159 Fr. 283 C->A 224 A->T 245 Stop at 262 138 G->T 165 Stop at 178 216 del 158 Stop at 167 258 Stop at 291 155 C->A 168 Ins 318 C->T 154 Stop at 167 266 Stop at 271 167 C->A 169 del 119 G->C/C-> 283 Stop at 304 284 Stop at 344 174 A->G 195 T->G G/C->G 284 C->A 285 ins 181 G->T 191 C->T 344 T->G 176 G->T/C->T 290 ins 241 T->A 152 Ins 216 Stop at 246 47 C->T/C->T 294 Stop at 344 305 A->T 168 A->C 240 G->C 151 C->T/C->T 308 Stop at 344 273 C->A 209 G->C 306 Stop at 344 88 C->T/C->T 49 Stop at 50 219 C->T 209 G->A 210 A->T 71 C->A 254 Stop at 260 251 C->G 214 T->G 198 A->T 162 T->C 301 Stop at 305 233 C->G 166 Fr. 273 Fr. 180 A->G 311 Stop at 344 215 Stop at 265 Stop at 344 138 del 150 Stop at 163 320 Stop at 344 246 163 C->G 171 Stop at 173 218 Stop at 219 324 Stop at 344 216 Ins 182 T->A 174 Ins 218 ins 244 Stop at 263 344 T->C 211 Ins 209 A->C 228 C->A 265 C->A 213 G->C 236 T->A 208 Stop at 215 248 Fr. 222 Stop at 246 82 C->T 267 C->T 285 Stop at 304 282 G->A/G->T 296 C->A 151 del 148 G->A 286 G->C 61 A->T 205 ins 180 G->A 133 Stop at 169 130 C->A 75 T->C 77 Fr. 337 G->A 174 Stop at 179 201 Fr. 76 G->A 86 C->T 281 A->T 239 A->C 330 Stop at 344 295 C->G 112 C->T 133 T->G 244 Fr. 131 Stop at 148 340 G->A 320 ins 236 del 271 G->T 236 Stop at 239 144 A->G 125 G->T 306 G->C 278 Fr. 236 Ins 126 T->G 266 Fr. 227 T->A 171 G->A 238 del 234 T->C/C->G 135 Stop at 148 138 G->C 229 G->A 313 Stop at 334 165 Fr. 316 Stop at 336 178 Stop at 161 C->G/C->A 240 Stop at 262 101 A->T 234 T->A/C->A 246 168 C->A 266 del 254 Stop at 344 233 C->T/A->C 213 C->T/A->G 241 T->C 276 Stop at 344 217 G->A/G->A 206 T->A/G->T 191 Stop at 154 Ins 166 Ins 169 Fr. 226 C->A 207 177 C->T/C->T 150 Stop at 169 167 A->T 302 G->C 236 A->G 202 G->C 222 C->A 148 A->C 260 C->G 196 G->C 250 C->T/C->G 218 T->A 155 Stop at 180 220 A->G/T->A 156 G->A 261 A->T 228 C->T 195 del 233 Stop at 246 339 G->T 303 G->C 141 C->A 248 del 325 G->C 166 C->G 182 G->A 221 G->C 275 T->A 185 A->T 184 Stop at 177 C->A 226 C->T 230 Stop at 239 186 Stop at 208 246 271 G->C 215 T->C 303 Stop at 344 184 Stop at 185 279 Stop at 292 A->T 85 C->T 279 del 187 Stop at 208 344 300 C->T 89 C->T 153 Fr. 234 C->T 140 C->T 319 A->T 101 A->G 235 A->C 235 Stop at 239 282 C->A 195 Fr. 132 A->T/A->G 82 Stop at 145 141 Stop at 169 162 T->A 269 G->C 160 G->A 196 Fr. 264 T->C 251 C->T 172 T->G 46 C->T 275 ins 76 A->T 241 Stop at 35 G->C 122 G->T 82 ins 96 T->C 246 90 Fr. 108 G->A 273 T->G 308 C->A 248 G->A/G->A 135 C->A 103 C->T 276 Stop at 305 96 C->G 362 Stop at 131 A->T 281 G->C/A-> 254 A->G 206 G->A 369 155 del G/C->G 225 T->G 241 Ins 81 C->T 228 Stop at 239 105 C->T 246 G->T 111 T->G 224 G->A/G->A 292 Stop at 305 273 C->T/G->A 93 G->A 238 Ins 197 T->G 387 del 140 Stop at 148 254 C->A 347 C->G 301 C->T 231 A->G 270 Stop at 337 294 Ins 154 G->T/C->T 157 G->C 268 A->C 190 Stop at 195 139 Fr. 212 T->G 282 G->A 179 T->C 275 Stop at 341 356 G->C 260 C->A 276 C->A 232 A->T 143 Stop at 148 182 G->C 123 Stop at 148 250 C->T/C->T 134 T->G 107 C->G 73 Stop at 122 146 T->G 279 G->T 274 G->A 194 Stop at 206 76 C->G 73 T->A 219 C->T/C->T 243 Stop at 246 271 Stop at 343 150 Stop at 165 165 G->T 152 C->T/C->T 131 A->C 254 del 214 Stop at 220 313 G->A 157 C->T 255 Stop at 262 130 ins 158 C->T 322 A->C 133 A->T 222 C->T/C->T 323 Stop at 340 289 Ins 195 C->T 291 G->T 192 A->T 333 G->A 369 del 203 G->A 232 C->G 188 G->A 224 Fr. 199 A->G 277 T->C 176 T->C 227 T->C 327 T->C 247 C->T 135 C->T 211 C->T 149 C->T 142 C->T/C->T 138 C->T/C->T 178 C->T 241 C->T/C->T 130 C->T 127 C->T/C->T 135 G->A 211 A->G 286 Fr. 168 Fr. 175 C->A 381 Fr. 263 Fr. 292 A->G

The mutations observed most frequently in cancerous pathologies are presented in Table 4 below.

TABLE 4 Position Wild p53 SEQ ID NO. 1 R273H GAGGTGCGTGTTTGTGC SEQ ID NO. 61 R248Q gcaTgaaccggaggcccaT SEQ ID NO. 62 R248W gcaTgaaccggaggcccaT SEQ ID NO. 63 R249S gcaTgaaccggaggcccaT SEQ ID NO. 64 G245S CTGCATGGGCGGCATGAAC SEQ ID NO. 65 R282W TGGGAGAGACCGGCGCACA SEQ ID NO. 66 R175H TGTGAGGCACTGCCCCCAC SEQ ID NO. 67 C242S TAACAGTTCCTGCATGGGCG SEQ ID NO. 68 Postion Mutated p53 R273H GAGGTGCATGTTTGTGC SEQ ID NO. 69 R248Q gcaTgaacCAgaggcccaT SEQ ID NO. 70 R248W GCATGAACTGGAGGCCAT SEQ ID NO. 71 R249S gcaTgaaccggagTcccaT SEQ ID NO. 72 G245S CTGCATGGGCAGAGCATGAAC SEQ ID NO. 73 R282W TGGGAGAGACTGGCGCACA SEQ ID NO. 74 R175H TGTGAGGCGCTGCCCCCAC SEQ ID NO. 75 C242S TAACAGTTCCTCCATGGGCG SEQ ID NO. 76

Thus, the oligonucleotides may be complementary to a target sequence belonging to the mutated gene of p53 carrying at least one of the mutations presented in Table 3, and most particularly at least one of the mutations of Table 4 above.

These oligonucleotides are capable of discriminating in an effective manner between the wild form and the mutated form of p3. The strategy is to block the expression of the mutated form to reactivate the wild form and induce in the cells a programmed death process for which the wild form is indispensable and/or to block any other process induced by the wild form of p53. Moreover, this discrimination capacity of the oligonucleotides of the invention makes it possible to not touch the cancerous cells and to spare the normal cells, which do not express this mutated form of p53.

We also provide for the treatment or prevention of diseases induced by an inactivation of protein p53 particularly the cancers resulting from the expression of mutated p53 and the cancers resulting from the expression of inhibitory genes of p53. We invention also provide for the prevention of the appearance of cancers in subjects expressing a mutated form of p53 as in the case of Li-Fraumeni cancer syndrome.

p53 can be inactivated via many distinct mechanisms. For example, in the majority of cervical cancers p53 is inactivated by E6, a protein coded by the human papilloma virus. E6 leads to the ubiquitinylation of p53 which leads to its degradation by the proteasome. In this case, the expression of p53 can be restored by inhibition of the expression of the protein E6. The aspects of this invention also relates to oligonucleotides presenting a sequence complementary to a specific polynucleotide sequence of the gene of the protein E6 of HPV. The sequence of the gene of the protein E6 of HPV is given in FIG. 6A as well as in the attached sequence listings as SEQ ID NO. 2.

As previously indicated, one strategy has as its goal to block using RNAi the expression of the androgen receptor in carcinomas. The sequence of the androgen receptor is given in the attached sequence listings as SEQ ID NO. 77. In order to treat carcinomas before they became resistant or to treat those that had become resistant by amplification of the receptor without mutation, siRNA homologous to a region for which no mutation had been described in the data banks of mutations of the androgen receptors (indicated as siRNA AR) were used. In order to treat specifically the prostate carcinomas that had become androgen resistant by mutation, a sequencing of the mRNA coding for the receptor was performed in the patient's cells in order to devise a specific sequence of the mutation, making it possible to treat the patient without consequence for the normal cells. An example is presented for the use of siRNA recognizing specifically the mutation of the androgen receptor present in the cell line LNCaP (siRNA LNCaP). Consequently, one aspect relates to oligonucleotides substantially complementary to a target sequence belonging to a DNA or messenger RNA molecule coding the mutated or nonmutated androgen receptor. For example, the androgen receptor bearing at least one of the mutations presented in Table 5. These oligonucleotides are specific to the androgen receptor and are useful for treating or preventing androgen-dependent diseases, such as, e.g., prostate cancer.

TABLE 5 Ac- Pathogenicity Position Change Exon 1 Androgen Binding ces- CpG Amino tracts Ther- sion Mutation Exon prov- hot acid Amino acid Poly Poly mo- Sex of External Family # Phenotype type Domain en spot Base Base Gln # Gly # Bmax Kd k labile Comments rearing Genitalia history Reference 0001 PAIS Substitute 1 * 002 Glu

 Lys high 20-50% reduction in Male Ambiguous pos Choong et al; J Clin Nterm 4 GAA

 AAA mutant protein Invest. 98: 1423-1431, 1996 0002 CAIS Insertion 1 051 Gly

 0 zero 1 nt insertion causing Female Normal pos Bruggenwirth et al; J Nterm GGC

 +C frameshift & stop in Steroid Biochem Mol Codon 180 Biol 58: 569-675, 1996 0003 Prostate Substitute 1 054 Leu

 Ser Also Phe891Leu Male Normal Tilley et al; Clinical cancer Nterm 523 TTG

 TCG (TTT to CTT) mut. Cancer Res. 2: 277-285, Somatic mutation 1996 0004 Laryngeal Deletion 1 057

30 nt. deletion Male Normal Urushibata et al; 10th. cancer Nterm

Somatic mutation Int. Cong. Endocrinol Abstr. P3-706, 1996 0005 Prostate Substitute 1 057 Leu

 Gln Somatic mutation Male Normal Tilley et al; Clinical cancer Nterm 532 CTG

 CAG Cancer Res. 2: 277-285, 1996 0411 Mental Deletion 1 058

8 normal normal 3 affected siblings - Male Normal pos Kooy et al; Am J Med Retard. Nterm

normal CAG = 23 Genet. 85: 389-393. 1999 0006 Kennedy Insertion 1 058-078

>40 Expansion of Male Normal LaSpada et al; Nature Syndrome Nterm

polyglutamine repeat 352: 77, 1991 0007 Prostate Deletion 1 058-078

18 Contraction of poly Male Normal Schoenberg et al; Bioch. cancer Nterm

Gln repeats (24 to 18) & Biophys Res Comm Somatic mutation 198: 74-80 1994 0324 Prostate Deletion 1 058-078

22 Deletion of 1polyGln Male Normal Watanabe et al; Jpn J cancer Nterm

repeat (23-22) Clin Oncol 27: 389-393, Somatic mutation 1997 0325 Prostate Insertion 1 058-078

22 Insertion of 1polyGln Male Normal Watanabe et al; Jpn J cancer Nterm

repeat (21-22) in 2 Clin Oncol 27: 389-393, diff patients. Som mut 1997 0495 Prostate Deletion 1 058-078

18 Contraction of poly Male Normal Wallin et al; J Pathology cancer Nterm

Gln repeats (20 to 18) 189: 559-653, 1999 Somatic mutation 0008 CAIS Substitute 1 * 060 Gln

 Stop low normal high Normal upregulation. Female Normal neg Zoppi et al; J Clin Inv Nterm 540 CAG

 TAG 19: 1105, 1993 0409 CAIS Insertion 1 060 Gln

 Gln either 1nt. insert or Female Normal pos Zhu et al; J Clin or Nterm 542 CAG

 CAAG 2nt. del. -frameshift Endocrinol & metab 84: deletion & stop in codon 80 1590-1594, 1999 0009 Prostate Substitute 1 064 Gln

 Arg Also Leu830Pro Male Normal Tilley et al; Clinical cancer Nterm 550 CAG

 CGG (CTT to CCT) mut. Cancer Res. 2: 277-285, Somatic mutation 1996 0416 CAIS Insertion 1 085 Gln

 Gln 25 zero 1nt. insertion causing Female Normal Gottlieb et al; Hum Nterm 617 CAG

 CAAG frameshift and stop in Mutat. 14: 527-539, codon 91 1999 0529 CWR22R Substitute 1 91 Glu

 Asp 27 19 AR indep. + Leu57Gln Male Normal Chelnski et al. The Prost. CA Nterm 635

& His 874Tyr mut. + Prostate 47: 66-75, 2001 Cell line Duplication of exon 3 0417 CAIS Deletion 1 102 Pro

 Pro 12 25 zero 1 nt. deletion causing Female Normal neg Gottlieb et al; Hum Nterm 668 CCΔC

 CCG frameshift and stop in Mutat. 14: 527-539, codon 172 1999 0010 Prostate Substitute 1 112 Gln

 His AlsoTrp798Stop Male Normal Tilley et al; Clinical cancer Nterm 698 CAG

 CAT (TGG to TGA) mut. Cancer Res. 2: 277-285, Somatic mutation 1996 0418 CAIS Substitute 1 113 Gln

 Stop 25 27 Female Normal Gottlieb et al; Hum Nterm 699 CAA

 TAA Mutat. 14: 527-539, 1999 0417 CAIS Deletion 1 125 Pro

 Pro 23 24 zero 1nt. deletion causing Female Normal Gottlieb et al; Hum Nterm 738 CCΔC

 CCG frameshift and stop at Mutat. 14: 527-539, codon 172 1999 0011 CAIS Deletion 1 127 Arg

 Arg zero 1 nt deletion causing Female Normal neg Batch et al; Hum Mol Nterm 743 AGΔA

 AGG frameshift & stop in Genet Codon 172 1: 497, 1992 0436 CAIS Deletion 1 127 Arg

 Arg 1 nt deletion causing Female Normal Ahmed et al; J Clin Nterm 743 AGΔA

 AGG frameshift & stop in Endocrinol & Metab 85: Codon 172 658-665, 2000 0012 CAIS Deletion 1 140

Deletion of Codons Female Normal Hiort et al; Am J Med Nterm

140-148 Stop in Genet. 63: 218-222, Codon 154 1996 0516 CAIS Substitute 1 153 Glu

 Stop Female Normal Copelli et al; Asian J Nterm 819 GAG

 TAG Androl 1: 73-77, 1999 0523 CAIS Substitute 1 153 Glu

 Stop Female Normal Gacobini et al. Hum Nterm 819 GAG

 TAG Genet. 108; 176, 2001 0013 CAIS Substitute 1 172 Leu

 Stop Female Normal Hiort et al; Am J Med Nterm 876 TTA

 TGA Genet. 63: 218-222, 1996 0316 PAIS Substitute 1 172 Leu

 Stop low normal Somatic mosaic mut. Female Ambiguous Holterhus et al; J Clin Nterm 876 TTA

 TGA causes partial Endocrinol. 82: 3584-3589, virulization 1997 0420 CAIS Substitute 1 172 Leu

 Stop 26 24 zero Female Normal neg Gottlieb et al; Hum Nterm 876 TTA

 TGA Mutat. 14: 527-539, 1999 0014 Prostate Substitute 1 180 Lys

 Arg Somatic mutation Male Normal Tilley et al; Clinical cancer Nterm 911 AAA

 AGA Cancer Res. 2: 277-285, 1996 0319 CAIS Substitute 1 194 Gln

 Arg Also 1 nt deletion in Female Normal Komori et al; J Nterm 943 CAA

 CGA Codon 597 causing Obstetrics & Gynocol. stop 23: 277-81, 1997 0551 Prostate Substitute 1 198 Glu

 Gly Treated with Male Normal Taplin et al; 37th cancer Nterm 955 GAA

 GGA bicalutamide - somatic meeting ASCO 20: mutation Abstr, 1738 2001 0015 CAIS Insertion 1 202 Glu

 0 zero 4 nt insertion causing Female Normal neg Batch et al; Hum Mol Nterm

frameshift & stop in Genet 1: 497, 1992 Codon 232 0549 Prostate Substitute 1 202 Glu

 Glu Treated with Male Normal Taplin et al; 37th cancer Nterm 968 GAA

 GAG bicalutamide - silent meeting ASCO 20: mutation- somat. mut. Abstr, 1738 2001 0395 Normal Substitute 1 205 Ser

 Arg Homosexual Male Normal Macke et al; Am J Nterm 977 AGC

 AGG individual Human Genetics 53: 844-852, 1993 0437 CAIS Deletion 1 208 Arg

 Lys zero Frameshift & stop in Female Normal Ahmed et al; J Clin Nterm 985 AΔGA

 AAG codon 232 ? Endocrinol & Metab 85: 658-665, 2000 0376 MAIS Substitute 1 210 Arg

 Arg Male Normal Wang et al; Clinical Nterm 992 AGG

 AGA Genetics 54; 185-192, 1998 0328 Normal Substitute 1 211 Glu

 Glu Silent mutation - Male Normal Batch et al; Hum Mol Nterm 995 GAG

 GAA polymorphism Genet 1: 497, 1992 detected in 8% popul. 0329 Normal Substitute 1 211 Glu

 Glu Silent mut. Male Normal Hiort et al; Eur J Pediatr Nterm 995 GAG

 GAA polymorph -detected in 153: 317-321, 1994 14% of X chromosomes 0330 Normal Substitute 1 211 Glu

 Glu Silent mutation Male Normal Lu et al; Clinical Nterm 995 GAG

 GAA polymorphism Genetics 49: 323-324. 1996 0377 Normal Substitute 1 211 Glu

 Glu Silent mutation Male Normal Wang et al; Clinical Nterm 995 GAG

 GAA polymorphism Genetics 54: 185-192, 1998 0396 Normal Substitute 1 211 Glu

 Glu Silent mut. polymorph Male Normal Macke et al; Am J Nterm 995 GAG

 GAA detected in 10% of X Human Genetics 53: chromosomes 844-852, 1993 0378 MAIS Substitute 1 211 Glu

 Glu Silent mutation Male Normal Wang et al; Clinical Nterm 995 GAG

 GAA polymorphism - 4 Genetics 54: 185-192, patients with infertility 1998 0421 CAIS Substitute 1 211 Glu

 Glu 22 24 v low Silent mutation - Female Normal Gottlieb et al; Hum Nterm 995 GAG

 GAA negligible level of Mutat. 14: 527-539, mRNA & hAR 1999 0422 CAIS Substitute 1 211 Glu

 Glu 21 23 normal normal Silent mutation - Female Normal neg Gottlieb et al; Hum Nterm 995 GAG

 GAA Mutat. 14: 527-539, 1999 0423 PAIS Substitute 1 211 Glu

 Glu 23 24 v low Silent mutation - Male Ambiguous Gottlieb et al; Hum Nterm 995 GAG

 GAA Mutat. 14: 527-539, 1999 0424 PAIS Substitute 1 211 Glu

 Glu 19 24 normal high Silent mutation - Male Ambiguous pos Gottlieb et at; Hum Nterm 995 GAG

 GAA Mutat. 14: 527-539, 1999 0425 MAIS Substitute 1 211 Glu

 Glu 20 16 normal high Silent mutation - Male Normal Gottlieb et al; Hum Nterm 995 GAG

 GAA Mutat. 14: 527-539, 1999 0379 MAIS Substitute 1 * 214 Gly

 Arg 27 23 normal normal norm servere oligospermia- Male Normal Wang et al; Clinical Nterm 1005 GGG

 AGG 20% lower Genetics 54: 185-192, transactivation 1998 0380 Normal Substitute 1 214 Gly

 Arg Male Normal Wang et al; Clinical Nterm 1005 GGG

 AGG Genetics 54: 185-192, 1998 0016 CAIS Insertion 1 215 Ala

 Gly 1 nt Insertion Female Normal neg Hiort et al; Hum Mol Nterm GCT

 GGCT causing frameshift & Genet stop in Codon 232 3: 1163-1166 1994 0548 Prostate Substitute 1 222 Asn

 Asp Treated with flutamide Male Normal Taplin et al; 37th cancer Nterm 1026 AAT

 GAT also Thr877Ala - meeting ASCO 20: somatic mutation Abstr, 1738 2001 0531 MAIS Substitute 1 * 233 Asn

 Lys normal * Azospermia - Male Normal Giwercman et al. Clin Nterm 1061 AAC

transactivation 46% of Endocrinol 54: 827-834, wt 2001 0350 CAIS Substitute 1 * 255 Leu

 Pro * Also Gly820Ala mut. Female Normal Tanaka et al; Gynocol Nterm 1126 CTG

 CCG Extra mutation causes Endocrinol 12: 75-82, greater thermolability 1998 0017 Prostate Substitute 1 266 Met

 Thr Also Leu574Pro Male Normal Tilley et al; Clinical cancer Nterm 1149 ATG

 ACG (CTG to CCC) mut. Cancer Res. 2: 277-285, Somatic mutation 1996 0018 Prostate Substitute 1 269 Pro

 Ser Somatic mutation Male Normal Tilley et al; Clinical cancer Nterm 1167 CCA

 TCA Cancer Res. 2: 277-285, 1996 0019 CAIS Deletion 1 272 Gly

 Gly zero 1 nt deletion causing Female Normal Bruggenwirth et al; J Nterm 1178 GGΔA

 GGG frameshift & stop in Steroid Biochem Mol Codon 301 Biol 58: 569-575, 1996 0556 Prostate Substitute 1 296 Ser

 Arg Poor differentiation of Male Normal Yu et al; Sheng Wu Hua cancer Nterm 1250 AGC

 AGA CaP. Germline Xue 32: 459-462, 2000 mutation ? 0550 Prostate Substitute 1 334 Ser

 Pro Treated with flutamide Male Normal Taplin et al; 37th cancer Nterm 1359 TCC

 CCC somatic mutation meeting ASCO 20: Abstr, 1738 2001 0398 Prostate Substitute 1 340 Pro

 Leu Somatic mutation Male Normal Castagnaro et al; Verh. cancer Nterm 1381 CCG

 CTG Stage 3 tumor Dtsch. Ges. Path. 77: 119-123, 1993 0020 CAIS Substitute 1 353 Glu

 Stop 21 23 low low specific binding Female Normal neg Gottlieb et al; Hum Nterm 1419 GAG

 TAG with MB only-mRNA Mutat. 14: 527-539, <20% 1999 0021 CAIS Substitute 1 371 Gly

 Stop Somatic instability of Female Normal pos Davies et al; Clinical Nterm 1474 GGA

 TGA polyglycine tract Endocrinology 43: 69-77, 1995 0338 MAIS Substitute 1 * 390 Pro

 Ser Oligospermia Male Normal Hiort et al; J Clin Nterm 1530 CCG

 TCG Endocrinol & Metab 85: 2810-2815, 2000 0504 MAIS Substitute 1 * 390 Pro

 Ser Oligospermia Male Normal Hiort et al; J Clin Nterm 1530 CCG

 TCG Endocrinol & Metab 85: 2810-2815, 2000 0547 Prostate Substitute 1 390 Pro

 Leu Treated with flutamide Male Normal Taplin et al; 37th cancer Nterm 1531 CCG

 CTG also Asn756Asp- meeting ASCO 20: somatic mutation Abstr, 1738 2001 0022 CAIS Substitute 1 390 Pro

 Arg 20 24 zero + substs. Glu211Glu Female Normal pos Gottlieb et al; Hum Nterm 1531 CCG

 CGG GAGtoGAA&Gln443 Mutat. 14: 527-539, Arg(CAGtoCGG) 1999 0426 CAIS Substitute 1 403 Gln

 Stop 28 23 zero mRNA <20% Female Normal Gottlieb et al; Hum Nterm 1569 CAG

 TAG Mutat. 14: 527-539, 1999 0438 CAIS Deletion 1 461 Gly

 Gly zero 1 nt. deletion causing Female Normal Ahmed et al; J Clin Nterm 1735 GGΔC

 GGG frameshift Endocrinol & Metab 85: 658-665, 2000 0410 CAIS Deletion 1 473 Glu

 Gly 24 22 2 nt. del causing Female Normal Thiele et al; J Clin Nterm 1779 GΔAG

 GGC frameshift & stop in Endocrinol & Metab 84: cod 499-mRNA 50% 1751-1753, 1999 0427 CAIS Deletion 1 473 Glu

 Gly 26 26 zero 2 nt. del causing Female Normal Gottlieb et al; Hum Nterm 1779 GΔAG

 GGC frameshift & stop in Mutat. 14: 527-539, cod 499-mRNA 50% 1999 0024 CAIS Substitute 1 480 Tyr

 Stop 15 15 zero Normal 110 KD AR Female Normal Gottlieb et al; Hum Nterm 1802 TAC

 TAA produced at 25% of Mutat. 14: 527-539, normal level 1999 0546 CAIS Deletion 1 487 Gln

 Stop Female Normal Boehmer et al; J Clin Nterm 1821 CAG

 TAG Endocrinol & Metab 86: 4151-4160, 2001 0439 CAIS Deletion 1 488 Gly

 0 low 5 nt. deletion causing Female Normal Ahmed et al; J Clin Nterm 1824

frameshift Endocrinol & Metab 85: 658-665, 2000 0440 CAIS Substitute 1 491 Gly

 Ser low low Female Normal Ahmed et al; J Clin Nterm 1833 GGC

 AGC Endocrinol & Metab 85: 658-665, 2000 0025 CAIS Substitute 1 502 Trp

 Stop Female Normal pos Bruggenwirth et al; J Nterm 1867 TGG

 TAG Steroid Biochem Mol Biol 58: 569-575, 1996 0339 MAIS Substitute 1 * 511 Val

 Val Oligospermia caused Male Normal Hiort et al; 80th US Nterm 1895 GTG

 GTA by silent mutation Endo Soc Meeting, Abstr P2-38, 1998 0026 Prostate Substitute 1 528 Asp

 Gly Somatic mutation Male Normal Tilley et al; Chemical cancer Nterm 1945 GAT

 GGT Cancer Res. 2: 277-285, 1996 0027 CAIS Substitute 1 534 Tyr

 Stop zero Female Normal neg McPhaul et al; FASEB J Nterm 1964 TAC

 TAG 5: 2910-15, 1991 0028 CAIS and Deletion 1-8

zero Termini not yet Female Normal neg Trifiro et al; Mol Cell mental

defined Endocrinol 75: 37-47, retardation 1991 0029 CAIS Deletion 1-8

zero Female Normal pos Quigley et al; J Clin

Endocrinol Metab 74: 927, 1992 0030 CAIS Deletion 1-8

zero Female Normal pos Hiort et al; Am J Med

Genet. 63: 218-22, 1996 0435 CAIS Deletion 1-8

zero Female Normal Ahmed et al; J Clin

Endocrinol & Metab 85: 658-665, 2000 0031 CAIS Deletion 2

Female Normal Quigley et al; J Cell

Biochem Suppl 16C; Abstr L323, 1992 0441 CAIS Duplicat 2

Duplication of exon 2 Female Normal Ahmed et al; J Clin

Endocrinol & Metab 85: 658-665, 2000 0032 PAIS Substitute 2 547 Leu

 Phe low high Also has Trp741Cys Male Ambiguous pos Karl et al; 76th US 2003 TTG

 TTT (TGG to TGT) Endo Soc Meeting, mutation Abstr 1735, 1994 0357 Prostate Deletion 2 547 Leu

 Leu Frameshift - somatic Male Normal Takahashi et al; Cancer cancer 2003 TTΔG

 TTC mutation Research 55: 1621-1624, 1995 0033 MAIS Substitute 2 548 Pro

 Ser Distal hypospadia, Male Near-normal pos Sutherland et al; J of 2004 CCC

 TCC variable penetrance in male Urology 156: 828-831, family members 1996 0023 CAIS Duplicat 2

Duplication of 8 nt. # Female Normal Lumbroso et al; 10th Int 2011

2011-2018 frameshift Cong of Endocrinol, & stop in Codon 563 Abstr P1-182, 1996 0358 Prostate Deletion 2 554 Pro

 Pro Frameshift - somatic Male Normal Takahashi et al; Cancer cancer 2023 CCΔA

 CCC mutation Research 55: 1621-1624, 1995 0359 Prostate Deletion 2 554 Pro

 Pro Frameshift - somatic Male Normal Takahashi et al; Cancer cancer 2023 CCΔA

 CCC mutation Research 55: 1621-1624, 1995 0034 CAIS Substitute 2 * 559 Cys

 Tyr normal normal Female Normal neg Zoppi et al; Mol DBD 2038 TGC

 TAC Endocrinol 6: 409, 1992 0035 PAIS Substitute 2 568 Gly

 Trp normal normal Female Normal Lobaccaro et al; Clin DBD 2064 GGG

 TGG Endocrinol, 40: 297, 1994 0036 PAIS Substitute 2 568 Gly

 Val normal Severe hypospadia Male Ambiguous Allera et al: J Clin DBD 2065 GGG

 GTG Endocrinol & Metab 80: 2697-2699, 1995 0037 PAIS Substitute 2 568 Gly

 Val normal normal Chang et al; 73rd US DBD 2065-6 GGG

 GTT Endo Soc Meeting, Abstr 28, 1991 0545 PAIS Substitute 2 571 Tyr

 His 21 Male Ambiguous Boehmer et al; J Clin DBD 2073 TAT

 CAT Endocrinol & Metab 86: 4151-4160, 2001 0558 PAIS Substitute 2 571 Tyr

 His DHT therapy effective Male Ambiguous Foresta et al; Am J Med DBD 2073 TAT

 CAT Genet 107: 259-260, 2002 0332 CAIS Substitute 2 * 571 Tyr

 Cys Female Normal Komori et al; Arch DBD 2074 TAT

 TGT Gynecol & Obstetrics 261: 95-100, 1998 0038 CAIS Substitute 2 573 Ala

 Asp normal Defective DNA Female Normal neg Bruggenwirth et al; J DBD 2080 GCT

 GAT binding & Steroid Biochem Mol transactivation Biol 58: 569-575, 1996 0489 Prostate Substitute 2 575 Thr

 Ala Somatic Mutation Male Normal Marcelli et al; Cancer Cancer DBD 2085 ACA

 GCA Research 60: 944-949, 2000 0039 CAIS Substitute 2 * 576 Cys

 Arg normal normal Female Normal pos Zoppi et al; Mol DBD 2088 TGT

 CGT Endocrinol 6; 409, 1992 0040 CAIS Substitute 2 576 Cys

 Phe normal normal Female Normal Chang et al; 73rd US DBD 2089 TGT

 TTT Endo Soc Meeting, Abstr 28, 1991 0407 CAIS Substitute 2 576 Cys

 Phe Lack of DNA Female Normal Hooper et al; 81st US DBD 2089 TGT

 TTT binding - 17 members Endo Soc Meeting, of same family Abstr P2-145, 1999 0554 PAIS Substitute 2 * 577 Gly

 Arg normal normal high Alters affinity & Nguyen et al; Mol DBD 2091 GGA

 AGA selectivity of AR-ARE Endocrinol interactions 15: 1790-1802, 2001 0509 PAIS Substitute 2 * 578 Ser

 Thr normal partial tranactivation in Male Ambiguous Giwercman et al; DBD 2095 AGC

 ACC COS cells Hormone Research 53: 83-88, 2000 0041 CAIS Substitute 2 579 Cys

 Tyr Female Normal Sultan et al, J Steroid DBD 2098 TGC

 TAC Biochem & Mol Biol: 46 519, 1993 0042 CAIS Substitute 2 * 579 Cys

 Phe normal normal Reduced transcription Female Normal pos Imasaki et al; Mol & DBD 2098 TGC

 TTC & DNA binding Cell Endocrinol 120: 15-24, 1996 0043 CAIS Deletion 2 579 Cys

 Cys zero Single nt. deletion Female Normal Imai et al, Annals of DBD 2099 TGΔC

 TGA causing frameshift & Clin Biochem, 32: stop in Codon 619 482-486, 1995 0487 Prostate Substitute 2 580 Lys

 Arg Somatic mutation Male Normal Marcelli et al; Cancer Cancer DBD 2101 AAG

 AGG Research 60: 944-949, 2000 0044 CAIS Substitute 2 * 581 Val

 Phe normal normal Female Normal Lumbroso et al; Fertil DBD 2103 GTC

 TTC Steril, 60: 814, 1993 0045 CAIS Deletion 2 * 582 Phe

 0 22 23 low normal 3 nt. del - Phe 582 del Female Normal neg Beitel et al; Hum Mol DBD 2104-6 GΔTCTT

 GTC 2nt. from 581, Int. Genet, 3: 21, 1994 582. 581 still Val 0442 CAIS Deletion 2 582 Phe

 0 normal normal 3 nt. del - of Phe Female Normal Ahmed et al; J Clin DBD 2106-8 TTC

Endocrinol & Metab 85: 658-665, 2000 0047 PAIS Substitute 2 582 Phe

 Ser zero Female Ambiguous Hiort et al; Hum Mol DBD 2107 TTC

 TCC Genet 3: 1163-1166 1994 0046 PAIS Substitute 2 * 582 Phe

 Tyr normal normal Reduced transcription Female Ambiguous pos Imasaki et al; Mol & DBD 2107 TTC

 TAC & DNA binding Cell Endocrinol 120: 15-24, 1996 0048 CAIS Substitute 2 585 Arg

 Lys Female Normal Sultan et al; J Steroid DBD 2116 AGA

 AAA Biochem & Mol Biol: 46 519, 1993 0049 CAIS Deletion 2-8

zero Similar 2-8 deletion Female Normal Jakubiczka et al; Human

in 2 different families Mutation 9: 57-61, 1997 0050 CAIS Deletion 3 *

high normal Produces internally Female Normal pos Quigley et al; Mol DBD

deleted protein Endocrinol 6: 1103, 1992 0051 CAIS Deletion 3

Female Normal pos Hiort et al; Am J Med DBD

Genet. 63: 218-22, 1996 0443 CAIS Deletion 3

zero Female Normal Ahmed et al; J Clin DBD

Endocrinol & Metab 85: 658-665, 2000 0444 CAIS Deletion 3

Female Normal Ahmed et al; J Clin DBD

Endocrinol & Metab 85: 658-665, 2000 0488 Prostate Substitute 3 586 Ala

 Val Somatic mutation Male Normal Marcelli et al; Cancer Cancer DBD 2119 GCC

 GTC Research 60: 944-949, 2000 0490 Prostate Substitute 3 587 Ala

 Ser Somatic mutation Male Normal Marcelli et al; Cancer Cancer DBD 2121 GCT

 TCT Research 60: 944-949, 2000 0052 CAIS Substitute 3 * 590 Lys

 Stop zero Female Normal Marcelli et al: Mol DBD 2130 AAA

 TAA Endocrinol 4: 1105, 1990 0053 PAIS Substitute 3 * * 596 Ala

 Thr normal normal Found in 2 unrelated Male Ambiguous pos Gast et al; Mol & Cell DBD 2148 GCC

 ACC fam. Abolishes Endocrinol 111: 93-98, dimerization 1995 0434 PAIS Substitute 3 * 596 Ala

 Thr normal normal Somatic mosaicism Male Ambiguous Holterhus et al; Pediatric DBD 2148 GCC

 ACC Res 46: 684-690, 1999 0510 PAIS Substitute 3 * * 596 Ala

 Thr normal partial transactivation Male Ambiguous Giwercman et al; DBD 2148 GCC

 ACC in COS cells Hormone Research 53: 83-88, 2000 0054 PAIS Substitute 3 * 597 Ser

 Gly normal normal High dissoc. rate. Female Ambiguous Zoppi et al; Mol DBD 2151 AGC

 GGC Also has Arg617Pro Endocrinol 6: 409, 1992 (CGG to CCG) mut. 0390 PAIS Substitute 3 597 Ser

 Thr Servere hypospadia Male Ambiguous Nordenskjold et al DBD 2152 AGC

 ACC and cryptorchidism Urological Res. 27: 49-55, 1999 0055 CAIS Substitute 3 601 Cys

 Phe Female Normal pos Baldazzi et al; Hum Mol DBD 2164 TGC

 TTC Genet 3: 1169-70 1994 0056 PAIS Substitute 3 604 Asp

 Tyr Male Ambiguous Hiort et al: Hum Mol DBD 2172 GAT

 TAT Genet 3: 1163-1166 1994 0057 CAIS Substitute 3 * 607 Arg

 Stop zero Female Normal Brown et al; Eur J DBD 2181 CGA

 TGA Pediatr (Suppl 2) 152; S62, 1993 0511 CAIS Substitute 3 * 607 Arg

 Stop zero Female Normal Giwercman et al; DBD 2181 CGA

 TGA Hormone Research 53: 83-88, 2000 0058 PAIS and Substitute 3 * 607 Arg

 Gln Male Ambiguous pos Wooster et al; Nat breast DBD 2182 CGA

 CAA Genet 2: 132, 1992 cancer 0059 PAIS Substitute 3 * * 607 Arg

 Gln normal normal Male Ambiguous pos Weidemann et al; Clin DBD 2182 CGA

 CAA Endocrinology 45: 733-739, 1996 0060 PAIS Substitute 3 * 607 Arg

 Gln Female Ambiguous Hiort et al; Am J Med DBD 2182 CGA

 CAA Genet. 63: 218-222, 1996 0347 PAIS Substitute 3 * 607 Arg

 Gln Patient successfully Male Ambiguous Weidemann et al; J Clin DBD 2182 CGA

 CAA treated with Endocrinol & Metab testostertone enanthate 83: 1173-1176, 1998 0393 PAIS Substitute 3 * 607 Arg

 Gln Germ cell tumour - in Female Normal Chen et al; Human DBD 2182 CGA

 CAA undescended testis Reproduction 14: 664-670, 1999 0412 CAIS Deletion 3 608

Mullerian ducts pres. Female Normal Chen et al; Fertility & DBD 2184

5 nt. del frameshift & Sterility 72: 170-173, stop in codon 619 1999 0061 PAIS Substitute 3 608 Arg

 Lys normal normal Male Ambiguous Saunders et al; Clin DBD 2185 AGG

 AAG Endocrinol 37: 214, 1992 0062 PAIS and Substitute 3 608 Arg

 Lys normal normal Male Ambiguous Lobaccaro et al; Hum breast DBD 2185 AGG

 AAG Mol Genet, 2: 1799, cancer 1993 0322 PAIS Substitute 3 608 Arg

 Lys normal normal Defective nuclear Male Ambiguous Tincello et al; Clinical DBD 2185 AGG

 AAG localization Endocrinology 46: 497-506, 1997 0352 PAIS Substitute 3 608 Arg

 Lys Male Ambiguous pos Hiort et al; J Pediatrics DBD 2185 AGG

 AAG 132: 939-943, 1998 0481 PAIS Substitute 3 608 Arg

 Lys normal high Ahmed et al; J Clin DBD 2185 AGG

 AAG Endocrinol & Metab 85: 658-665, 2000 0063 PAIS Substitute 3 * 610 Asn

 Thr normal low * Male Ambiguous Weidemann et al; Clin DBD 2190 AAT

 ACT Endocrinology 45: 733-739, 1996 0496 CAIS Substitute 3 611 Cys

 Tyr Female Normal Mockel et al; Geburtshi. DBD 2193 TGT

 TAT und Frauen. 60: 232-234, 2000 0064 CAIS Deletion 3 * 615 Arg

 0 27 23 normal normal 3 nt. del - Arg615 del, Female Normal pos Beitel et al; Hum Mol DBD 2204-6  TGΔTCG

 TGT 1 nt. from 614, 2nt. Genet, 3: 21, 1994 615. 614 still Cys 0512 CAIS Substitute 3 * 615 Arg

 Gly no transactivation in Female Normal Giwercman et al; DBD 2205 CGT

 GGT COS cells Hormone Research 53: 83-88, 2000 0065 CAIS Substitute 3 * * 615 Arg

 His 25 23 low high Female Normal pos Beitel et al; Hum Mol DBD 2206 CGT

 CAT Genet, 3: 21, 1994 0066 CAIS Substitute 3 * * 615 Arg

 His normal normal Female Normal pos Mowszowicz et al; Mol DBD 2206 CGT

 CAT Endocrinol 7: 861-869, 1993 0067 CAIS Substitute 3 * * 615 Arg

 His Female Normal Brown et al; Eur J DBD 2206 CGT

 CAT Pediatr 152 (Suppl 2): S62, 1993 0068 CAIS Substitute 3 * * 615 Arg

 His Female Normal Ris-Stalpers et al; DBD 2206 CGT

 CAT Pediatr Res 36: 227, 1994 0348 CAIS Substitute 3 * 615 Arg

 His Female Normal Cabral et al; Brazilian J DBD 2206 CGT

 CAT Mol & Biol Res. 31: 775-778, 1998 0353 CAIS Substitute 3 * 615 Arg

 His Female Normal Hiort et al; J Pediatrics DBD 2206 CGT

 CAT 132: 939-943, 1998 0354 CAIS Substitute 3 * 615 Arg

 His Female Normal Hiort et al; J Pediatrics DBD 2206 CGT

 CAT 132: 939-943, 1998 0069 PAIS Substitute 3 * 615 Arg

 His Male Ambiguous Hiort et al; Am J Med DBD 2206 CGT

 CAT Genet. 63: 218-222, 1996 0070 PAIS Substitute 3 615 Arg

 Pro Male Ambiguous Hiort et al; Am J Med DBD 2206 CGT

 CCT Genet. 63: 218-222, 1996 0445 CAIS Substitute 3 615 Arg

 Pro normal high Female Normal Ahmed et al; J Clin DBD 2206 CGT

 CCT Endocrinol & Metab 85: 658-665, 2000 0071 PAIS Substitute 3 * 616 Leu

 Arg normal normal Female Ambiguous pos De Bellis et al; J Clin DBD 2209 CTT

 CGT Endocrinol Metab, 78: 513, 1994 0072 CAIS Substitute 3 616 Leu

 Pro Female Normal Mebarki et al; 75th US DBD 2209 CTT

 CCT Endo Soc Meeting, Abstr 602, 1993 0073 CAIS Substitute 3 * 616 Leu

 Pro normal normal Female Normal Lobaccaro et al; Mol DBD 2209 CTT

 CCT Cell Endorinol, 5: 137-147, 1996 0074 PAIS Substitute 3 * 617 Arg

 Pro normal normal Female Ambiguous pos Marcelli et al; J Clin DBD 2212 CGG

 CCG Invest. 87: 1123, 1991 0075 PAIS Substitute 3 * 617 Arg

 Pro normal normal high Mutation also at 597 Female Normal Zoppi et al; Mol DBD 2212 CGG

 CCG Endocrinol 6: 409, 1992 0431 Prostate Substitute 3 * 619 Cys

 Tyr low high Inactive transcription Male Normal Nazereth et al; Mol cancer DBD 2218 TGT

 TAT Does not bind DNA Endocrinol 13: somatic mutation 2065-2075, 1999 0491 Prostate Substitute 3 619 Cys

 Tyr Somatic mutation Male Normal Marcelli et al; Cancer cancer DBD 2218 TGT

 TAT Research 60: 944-949, 2000 0076 CAIS Deletion 3-8

Female Normal Brown et al, Eur J

Pediatr (Suppl 2) 152: S62, 1993 0077 MAIS Deletion 4

Azoospermia Male Normal neg Aiken et al; Am J Obs LBD

& Gyn. 165: 1891-1894, 1991 0078 CAIS Deletion 4 *

13 nt deletion causing Female Normal pos Lobaccaro et al; Mol & LBD

frameshift and stop at Cellular Endocrinology codon 783 111: 21-8, 1995 0306 Prostate Substitute 4 629 Arg

 Gln 1 of 6 of hormone- Male Normal Wang et al; Japanese J cancer 2248 CGG

 CAG independent D2 of Urology 88: 550-556 patients-somatic mut 1997 0079 Prostate Substitute 4 630 Lys

 Thr Also Lys717Glu mut, Male Normal Tilley et al; Clinical cancer 2251 AAG

 ACG (AAGtoGAG) + silent Cancer Res. 2: 277-285, mut in 701. Som mut 1996 0400 CAIS Substitute 4 640 Gln

 Stop zero Female Normal Yaegashi et al; Tohoku J LBD 2280 CAG

 TAG of Exp Med 187: 263-272, 1999 0429 CAIS Substitute 4 640 Gln

 Stop zero also Trp751Stop mut, Female Normal Uehara et al; Am J Med LBD 2280 CAG

 TAG (TGGtoTGA) 47XXY Genet. 86: 107-111, Muts on both X's 1999 0080 PAIS Substitute 4 645 Ala

 Asp Male Ambiguous Hiort et al; Am J Med LBD 2296 GCT

 GAT Genet. 63: 218-222, 1996 0334 Normal Substitute 4 645 Ala

 Asp Male Normal Nordenskjold et al; LBD 2296 GCT

 GAT Human Mutation. 11: 339, 1998 0081 Prostate Substitute 4 647 Ser

 Asn +Gly724Asp, Leu880 Male Normal Taplin et al; New cancer LBD 2302 AGC

 AAC Gln & Ala896Thr.mut England J Med Somatic mutations 332: 1393-1398, 1995 0555 PAIS Substitute 4 653 Glu

 Lys 20 Also in family with Male Ambiguous Lundberg et al; J Clin LBD 2319 GAG

 AAG CAH with no Endocrinol & Metab 87: androgen insensitivity 2023-2028, 2002 0517 CAIS Substitute 4 657 Gln

 Stop Female Normal Chavez et al; Clin Genet LBD 2231 CAG

 TAG 59:: 185-188, 2001 0082 PAIS Substitute 4 664 Ile

 Asn 22 22 low norm Pinsky et al; Clin Inv LBD 2353 ATT

 AAT Med 15: 456, 1992 0083 Prostate Substitute 4 670 Gln

 Arg Also Ser791Pro Male Normal Tilley et al; Clinical cancer LBD 2371 CAG

 CGG (TCT to CCT) mut. Cancer Res. 2: 277-285, Somatic mutation 1996 0084 PAIS Substitute 4 671 Pro

 His Male Ambiguous Hiort et al; Am J Med LBD 2374 CCC

 CAC Genet. 63: 218-222, 1996 0085 Prostate Substitute 4 672 Ile

 Thr Somatic mutation Male Normal Tilley et al; Clinical cancer LBD 2377 ATC

 ACC Cancer Res. 2: 277-285, 1996 0086 CAIS Substitute 4 677 Leu

 Pro zero Female Normal pos Belsham et al; Human LBD 2392 CTG

 CCG Mutation 5: 28-33, 1995 0087 CAIS Substitute 4 681 Glu

 Lys Female Normal Hiort et al; J Clin LBD 2403 GAG

 AAG Endocrinol Metab 77: 262-266, 1993 0394 CAIS Substitute 4 681 Glu

 Lys Germ cell tumour in Female Normal Chen et al; Human LBD 2403 GAG

 AAG undescended testis Reproduction 14: 664-670, 1999 0534 PAIS Substitute 4 682 Pro

 Thr low Female Ambiguous Chavez et al; J Hum LBD 2406 CCA

 ACA Genet. 46: 560-565, 2001 0089 Prostate Substitute 4 683 Gly

 Ala Somatic mutation - Male Normal Koivisto et al; Cancer cancer LBD 2410 GGT

 GCT transactivation Research 57: 314-319, normal 1997 0090 CAIS Substitute 4 684 Val

 Ile zero Female Normal Mebarki et al; 75th US LBD 2412 GTA

 ATA Endo Soc Meeting, Abstr 602, 1993 0091 PAIS Substitute 4 686 Cys

 Arg Male Ambiguous Hiort et al; Am J Med LBD 2418 TGT

 CGT Genet. 63: 218-222, 1996 0092 PAIS Substitute 4 687 Ala

 Val Male Ambiguous Hiort et al; Am J Med LBD 2422 GCT

 GTT Genet. 63: 218-222, 1996 0093 CAIS Substitute 4 688 Gly

 Glu de novo mutation Female Normal neg Hiort et al; J Pediatrics LBD GGA

132: 939-943, 1998 0446 CAIS Substitute 4 688 Gly

 Stop zero Female Normal Ahmed et al; J Clin LBD 2424 GGA

 TGA Endocrinol & Metab 85: 658-665, 2000 0094 PAIS Deletion 4 690 Asp

 0 Schwartz et al; Horm LBD 2428-30 ACG

 0 Res 41: 117 Abstr 244, 1994 0095 CAIS Deletion 4 692 Asn

 0 normal high * Three nucleotide Female Normal Batch et al; Hum Mol LBD 2436-8  AAC

 0 deletion Genet 1: 497, 1992 0096 CAIS Substitute 4 * 695 Asp

 His low Female Normal neg Ris-Stalpers et at; Mol LBD 2445 GAC

 CAC Endocrinol 5: 1562, 1991 0097 CAIS Substitute 4 * * 695 Asp

 Asn normal normal high mutation found in two Female Normal pos Ris-Stalpers et at; Mol LBD 2445 GAC

 AAC unrelated families Endocrinol 5: 1562, 1991 0098 PAIS Substitute 4 * 695 Asp

 Asn de novo mutation Female Ambiguous Hiort et al; J Pediatrics LBD 2445 GAC

 AAC 132: 939-943, 1998 0335 CAIS Substitute 4 695 Asp

 Val 21 mtuation found in two Female Normal pos Dork et al; Human LBD 2446 GAC

 GTC siblings Mutation 11: 337-339, 1998 0447 CAIS Substitute 4 700 Leu

 Met Female Normal Ahmed et al; J Clin LBD 2460 TTG

 ATG Endocrinol & Metab 85: 658-665, 2000 0448 CAIS Substitute 4 701 Leu

 Phe Female Normal Ahmed et al; J Clin LBD 2463 CTC

 TTC Endocrinol & Metab 85: 658-665, 2000 0518 PAIS Substitute 4 701 Leu

 Ile Chavez et al; Clin Genet LBD 2463 CTC

 ATC 59:: 185-188, 2001 0099 Prostate Substitute 4 701 Leu

 His Somatic mutation Male Normal Suzuki et al; J Steroid cancer LBD 2464 CTC

 CAC Biochem Molec Biol 46: 759, 1993 0326 Prostate Substitute 4 701 Leu

 His Somatic mutation Male Normal Watanabe et al; Jpn J cancer LBD 2464 CTC

 CAC Clin Oncol 27: 389-393, 1997 0408 MDA Substitute 4 701 Leu

 His normal low Som. mut. Prostate Male Normal Zao et al; J of Urology PCa-Za LBD 2464 CTC

 CAC cancer cell line. Also 162: 2192-2199, 1999 has Thr877Ala 0100 CAIS Substitute 4 702 Ser

 Ala zero Female Normal Pinsky et al; Clin Inv LBD 2466 TCT

 GCT Med 15: 456, 1992 0101 PAIS Substitute 4 * 703 Ser

 Gly low high Male Ambiguous Radnayr et al; J of LBD 2469 AGC

 GGC Urology 158: 1553-1556, 1997 0449 CAIS Substitute 4 * 703 Ser

 Gly Female Normal Ahmed et al; J Clin LBD 2469 AGC

 GGC Endocrinol & Metab 85: 658-665, 2000 0559 CAIS Substitute 4 705 Asn

 Tyr Sister a carrier Female Normal Sills et al; Int J Mol LBD 2475 AAT

 TAT Med 9: 45-48, 2002 0102 CAIS Substitute 4 705 Asn

 Ser zero Female Normal Pinsky et al; Clin Inv LBD 2476 AAT

 AGT Med 15: 456, 1992 0103 CAIS Substitute 4 705 Asn

 Ser zero Female Normal DeBellis et al; Mol LBD 2476 AAT

 AGT Endocrinol 6: 1909-20, 1992 0104 CAIS Substitute 4 705 Asn

 Ser Mutation found in two Female Normal Quigley et al; Endocrine LBD 2476 AAT

 AGT unrelated families Reviews 16: 271, 1995 0482 PAIS Substitute 4 705 Asn

 Ser normal high Ahmed et al; J Clin LBD 2476 AAT

 AGT Endocrinol & Metab 85: 658-665, 2000 0105 CAIS Substitute 4 * 707 Leu

 Arg Female Normal Lumbroso et al; J Clin LBD 2482 CTG

 CGG Endo & Metab 81: 1984-1988, 1996 0106 PAIS Substitute 4 708 Gly

 Ala Male Ambiguous Hiort et al: Hum Mol LBD 2485 GGA

 GCA Genet 3: 1163-1166 1994 0314 PAIS Substitute 4 708 Gly

 Ala Servere hypospadias Male Ambiguous Albers et al; J of LBD 2485 GGA

 GCA Pediatrics 131: 388-392, 1997 0107 CAIS Substitute 4 708 Gly

 Val zero Male Ambiguous pos Auchus et al: 77th US LBD 2485 GGA

 GTA Endo Soc Meeting, Abstr P1-508 1995 0450 CAIS Substitute 4 710 ArG

 Thr zero Female Normal Ahmed et al; J Clin LBD 2491 AGA

 ACA Endocrinol & Metab 85: 658-665, 2000 0525 PAIS Substitute 4 * 711 Gln

 Glu v low altered AR specificity Female Ambiguous pos Lumbroso et al. 83rd LBD 2493 CAG

 GAG 2x increased affinity US Endo Soc Meeting, for E2. Abstr P2-29, 2001 0535 PAIS Substitute 4 * 711 Gln

 Glu normal Female Ambiguous pos Chavez et al; J Hum LBD 2493 CAG

 GAG Genet. 46: 560-565, 2001 0108 PAIS Substitute 4 * 712 Leu

 Phe normal high Phenotypic diversity Male Ambiguous pos Hiort et al; Am J Med LBD 2496 CTT

 GTT brother of 505& 506 Genet. 63: 218-222, Testost-induced norm. 1996 0505 PAIS Substitute 4 * 712 Leu

 Phe normal high Phenotypic diversity Male Ambiguous pos Hiort et al; J Clin LBD 2496 CTT

 GTT brother of 108& 506 Endocrinol & Metab 85: Testost-induced norm. 3245-3250, 2000 0506 PAIS Substitute 4 * 712 Leu

 Phe normal high Phenotypic diversity Male Ambiguous pos Hiort et al; J Clin LBD 2496 CTT

 GTT brother of 505& 108 Endocrinol & Metab 85: Testost-induced norm. 3245-3250, 2000 0507 PAIS Substitute 4 * 712 Leu

 Phe normal high Phenotypic diversity Male Ambiguous pos Hiort et al; J Clin LBD 2496 CTT

 GTT uncle of 108, 505, 506 Endocrinol & Metab 85: Testost-induced norm. 3245-3250, 2000 0109 Prostate Substitute 4 * * 715 Val

 Met normal Somatic mutation. Male Normal Culig et al; Mol cancer LBD 2507 GTG

 ATG Receptor showed a Endocrinol gain in function 7: 1541-1550 1993 0110 Prostate Substitute 4 * 715 Val

 Met normal Somatic mutation. Male Normal Bubley et al 87th Am cancer LBD 2507 GTG

 ATG Receptor showed a Assoc Cancer Res Meet gain in function Abstr. 1680, 1996 0111 CAIS Substitute 4 718 Trp

 Stop zero Female Normal pos Sai et al; Am J Hum LBD 2516 TGG

 TGA Genet 46: 1095, 1990 0112 Prostate Substitute 4 720 Lys

 Glu Somatic mutation- Male Normal Kleinerman et al; J of cancer LBD 2520 AAG

 GAG Bone metasteses of Urology 155: 624A, Prostate cancer 1996 0113 Prostate Substitute 4 721 AlA

 Thr Somatic mutation in Male Normal Taplin et al; New cancer LBD 2523 GCC

 ACC 20% of isolates in England J Med 332: initial cloning 1393-1398, 1995 0114 CAIS Substitute 4 722 Leu

 Phe Female Normal Hiort et al: Am J Med LBD 2526 TTG

Genet. 63: 218-222, 1996 0451 CAIS Substitute 4 723 Pro

 Ser normal high Female Normal Ahmed et al; J Clin LBD 2529 CCT

 TCT Endocrinol & Metab 85: 658-665, 2000 0452 CAIS Substitute 4 724 Gly

 Ser zero Female Normal Ahmed et al; J Clin LBD 2532 GGC

 AGC Endocrinol & Metab 85: 658-665, 2000 0453 CAIS Substitute 4 724 Gly

 Asp zero Female Normal Ahmed et al; J Clin LBD 2533 GGC

 GAC Endocrinol & Metab 85: 658-665, 2000 0115 CAIS Deletion 4-8

zero Female Normal Brown et al; Proc Natl LBD

Acad Sci 85: 8151, 1988 0116 CAIS Deletion 5

zero Affected aunt deleted Female Normal pos Maclean et al; J Clin LBD

for exons 6 and 7 Invest, 91: 1123, 1993 only. 0117 CAIS Substitute 5 Tyr

 Arg zero Female Normal Marcelli et al; 74th US LBD

Endo Soc Meetings: Abstr. 224, 1992 0118 PAIS Substitute 5 725 Phe

 Leu normal normal Quigley et al; Endocrin LBD 2535 TTC

 CTC Reviews 16: 271, 1995 0391 PAIS Substitute 5 725 Phe

 Leu Hypospadia and Male Ambiguous pos Nordenskjold et al LBD 2535 TTC

 CTC cryptorchidism Urological Res, 27: 49-55, 1999 0119 Prostate Substitute 5 * 726 ArG

 Leu normal normal Germ line mutation Male Normal pos Elo et al; J Clin cancer LBD 2539 CGC

 CTC present in offspring Endorinol Metab, 80: 3494-3500, 1995 0508 Prostate Substitute 5 * 726 ArG

 Leu Estimated that 2% of Male Normal pos Mononen et al; Cancer cancer LBD 2539 CGC

 CTC Finnish CAP patients Res 60: 6479-6481, have this mutation 2000 0120 MAIS Substitute 5 727 Asn

 Lys Oligospermia Male Normal Yong et al; Lancet, 344: LBD 2543 AAC

 AAG 826-827, 1994 0121 PAIS Substitute 5 728 Leu

 Ser low * McPhaul et al; J Clin LBD 2545 TTA

 TCA Inv, 90: 2097, 1992 0122 Prostate Substitute 5 * 730 Val

 Met Somatic mutation Male Normal Newmark et al; Proc Cancer LBD 2550 GTG

 ATG Natl AcadSci 89: 6319, 1992 0123 Prostate Substitute 5 * 730 Val

 Met Somatic mutation Male Normal Petersiel et al; Int J Cancer LBD 2550 GTG

 ATG Cancer 63: 544-550, 1995 0310 CAIS Substitute 5 732 Asp

 Asn 19 Female Normal Ko et al; J Reprod. LBD 2556 GAC

 AAC Med 42: 424-427, 1997 0125 CAIS Substitute 5 * 732 Asp

 Tyr high Female Normal Brown et al; 74th US LBD 2556 GAC

 TAC Endo Soc Meeting, Abstr 1506, 1992 0126 CAIS Substitute 5 732 Asp

 Tyr zero Female Normal Pinsky et al; Clin Inv LBD 2556 GAC

 TAC Med 15: 456, 1992 0127 CAIS Substitute 5 732 Asp

 Tyr Female Normal Ghirri and Brown; LBD 2556 GAC

 TAC Pediatr Res 33. Abstr 95, 1993 0124 CAIS Substitute 5 732 Asp

 Asn high Female Normal Brown et al; 74th US LBD 2556 GAC

 AAC Endo Soc Meeting, Abstr 1506, 1992 0128 PAIS Substitute 5 733 Gln

 His This patient was a Female Ambiguous neg Hiort et al; J Pediatrics LBD 2561 CAG

 CAT mosaic for wt. & mut. 132: 939-943, 1998 alleles-de novo mut. 0129 PAIS Substitute 5 737 Ile

 Thr low Quigley et al; Endocrin LBD 2571 ATT

 ACT Reviews 16: 271, 1995 0530 CAIS Substitute 5 * 739 Tyr

 Asp zero no transactivation in Female Normal Suzuki et al. Int. J LBD 2577 TAC

 GAC COS-1 cells Andrology 24: 183-188, 2001 0130 CAIS Substitute 5 * 741 Trp

 Arg low Female Normal neg Marcelli et al; J Clin LBD 2583 TGG

 CGG Invest 94: 1642-1650, 1994 0360 Prostate Substitute 5 741 Trp

 Stop Somatic mutation Male Normal Takahashi et al; Cancer cancer LBD 2584 TGG

 TAG Research 55: 1621-1624, 1995 0552 Prostate Substitute 5 741 Trp

 Cys Treated with Male Normal Taplin et al; 37th cancer LBD 2584 TGG

 TGG bicalutamide - somatic meeting ASCO 20: mutation Abstr. 1738 0131 PAIS Substitute 5 742 MeT

 Val high Ris-Stalpers et al; LBD 2586 ATG

 GTG Pediatric Res. 36: 227-234, 1994 0341 PAIS Substitute 5 742 MeT

 Val pos Melo et al; 80th US LBD 2586 ATG

 GTG Endo Soc Meeting Abstr P2-44, 1998 0132 PAIS Substitute 5 * 742 MeT

 Ile normal high * Female Ambiguous Batch et al; Hum Mol LBD 2588 ATG

 ATA Genet 1: 497, 1992 0519 CAIS Substitute 5 743 Gly

 Arg Female Normal Chavez et al; Clin Genet LBD 2589 GGG

 CGG 59:: 185-188, 2001 0133 PAIS Substitute 5 * 743 Gly

 Val low high Transcription only at Female Ambiguous Georget et al; J Clin LBD 2590 GGG

 GTG high conc of androgen Endocrinol & Metab 83: 3597-3603, 1998 0134 PAIS Substitute 5 743 Gly

 Val normal normal * Nakao et al; J Clin LBD 2590 GGG

 GTG Endocrinol Metab 77: 103-107, 1993 0414 CAIS Substitute 5 743 Gly

 Val zero de novo mutation Female Normal Lobaccaro et al; J LBD 2590 GGG

 GTG Steroid Biochem & Mol Biol. 44: 211-216, 1993 0536 CAIS Substitute 5 743 Gly

 Glu normal Female Normal Chavez et al; J Hum LBD 2590 CGG

 GAG Genet. 46: 560-56, 2001 0361 Prostate Deletion 5 743 Gly

 Gly Frameshift-somatic Male Normal Takahashi et al; Cancer cancer LBD 2591 GGΔG

 GGC mut.-separate tumor Research 55: in same indv. as 0362 1621-1624, 1995 0135 CAIS Substitute 5 744 Leu

 Phe Brinkmann et al; J LBD 2592 CTC

 TTC Steroid Biochem & Mol Biol 53: 443, 1995 0362 Prostate Substitute 5 744 Leu

 Phe Somatic mutation- Male Normal Takahashi et al; Cancer cancer LBD 2592 CTC

 TTC separate tumor in Research 55: same indv. as 0361 1621-1624, 1995 0136 PAIS Substitute 5 745 Met

 Thr zero Ris-Stalpers et al; LBD 2597 ATG

 ACG Pediatric Res 36: 227-234, 1994 0137 PAIS Substitute 5 746 Val

 Met Brown et al; 74th US LBD 2598 GTG

 ATG Endo Soc Meeting, Abstr 1506, 1992 0138 PAIS Substitute 5 746 Val

 Met Male Ambiguous Hiort et al; Am J Med LBD 2598 GTG

 ATG Genet. 63: 218-222 1996 0492 Prostate Substitute 5 748 Ala

 Thr Also Ser865Pro; Male Normal Marcelli et al; Cancer cancer LBD 2604 GCC

 ACC Gln867Stop and Research 60: 944-949, Gln919Arg; Som mut 2000 0139 PAIS Substitute 5 * 748 Ala

 Asp low high Abnormal dissociation Marcelli et al; J Clin LBD 2605 GCC

 GAC Invest 94: 1642-1650, 1994 0363 Prostate Substitute 5 748 Ala

 Val Somatic mutation Male Normal Takahashi et al; Cancer cancer LBD 2605 GCC

 GTC Research 55: 1621-1624, 1995 0140 CAIS Substitute 5 749 Met

 Val Female Normal pos DeBellis et al; Mol LBD 2607 ATG

 GTG Endocrinol 6: 1909-20, 1992 0141 CAIS Substitute 5 749 Met

 Val Female Normal pos Jakubiczka et al; Hum LBD 2607 ATG

 GTG Genet 90: 311-2, 1992 0483 PAIS Substitute 5 749 Met

 Val normal high Ahmed et al; J Clin LBD 2607 ATG

 GTG Endocrinol & Metab 85: 658-665, 2000 0364 Prostate Substitute 5 749 Met

 Ile Somatic mutation Male Normal Takahashi et al; Cancer cancer LBD 2609 ATG

 ATA Research 55: 1621-1624, 1995 0365 Prostate Substitute 5 750 Gly

 Ser Somatic mutation Male Normal Takahashi et al; Cancer cancer LBD 2610 GGC

 AGC Research 55: 1621-1624, 1995 0142 CAIS Substitute 5 * 750 Gly

 Asp vlow Mutation found in two Female Normal Bevan et al; J Steroid LBD 2611 GGC

 GAC unrelated patients Biochem Molec. Biol 61: 19-26, 1997 0143 CAIS Substitute 5 750 Gly

 Asp Female Normal Brown et al; 74th US LBD 2611 GGC

 GAC Endo Soc Meeting Abstr 1506, 1992 0144 CAIS Substitute 5 751 Trp

 Arg Female Normal Brinkmann et al; J LBD 2613 TGG

 AGG Steroid Biochem Mol Biol 53: 443, 1995 0366 Prostate Substitute 5 751 Trp

 Stop Somatic mutation Male Normal Takahashi et al; Cancer cancer LBD 2614 TGG

 TAG Research 55: 1621-1624, 1995 0367 Prostate Substitute 5 751 Trp

 Stop Somatic mutation Male Normal Takahashi et al; Cancer cancer LBD 2614 TGG

 TAG Research 55: 1621-1624, 1995 0368 Prostate Substitute 5 751 Trp

 Stop Somatic mutation Male Normal Takahashi et al; Cancer cancer LBD 2615 TGG

 TGA Research 55: 1621-1624, 1995 0401 CAIS Substitute 5 751 Trp

 Stop zero Female Normal Yaegashi et al; Tohoku J LBD 2615 TGG

 TGA of Exp Med 187: 263-272, 1999 0145 CAIS Substitute 5 * 752 Arg

 Stop zero Female Normal Pinsky et al; Clin Inv LBD 2616 CGA

 TGA Med 15: 456, 1992 0146 CAIS Substitute 5 * 752 Arg

 Stop Female Normal Brinkmann et al; J LBD 2616 CGA

 TGA Steroid Biochem Mol Biol 53: 443, 1995 0342 CAIS Substitute 5 * 752 Arg

 Stop In two different Female Normal Melo et al; 80th US LBD 2616 CGA

 TGA families Endo Soc Meeting Abstr P2-44, 1998 0402 CAIS Substitute 5 * 752 Arg

 Stop zero Female Normal Yaegashi et al; Tohoku J LBD 2616 CGA

 TGA of Exp Med 187: 263-272, 1999 0147 CAIS Substitute 5 * 752 Arg

 Gln zero Mutation found in two Female Normal Brown et al; 74th US LBD 2617 CGA

 CAA unrel. families. Endo Soc Meeting, Equivalent to tfm rat Abstr 1506, 1992 0148 CAIS Substitute 5 * 752 Arg

 Gln zero Equivalent to tfm rat Female Normal Evans; J Endocrinol 135 LBD 2617 CGA

 CAA Suppl, Abstr P26, 1992 0333 CAIS Substitute 5 * 752 Arg

 Gln Female Normal pos Komori et al; Arch LBD 2617 CGA

 AA Gynecol & Obstetrics 261: 95-100, 1998 0349 CAIS Substitute 5 * 752 Arg

 Gln Female Normal Cabral et al; Brazilian J LBD 2617 CGA

 CAA Med & Biol Res. 31: 775-758, 1998 0497 CAIS Substitute 5 * 752 Arg

 Gln Bilateral testicular Female Normal Sakai et al; International LBD 2617 CGA

 CAA tumors J of Urology 7: 390-392, 2000 0149 CAIS Substitute 5 754 Phe

 Val zero Female Normal Lobaccaro et al; Hum LBD 2622 TTC

 GTC Mol Genet 2: 1041-1043, 1993 0150 CAIS Substitute 5 754 Phe

 Val Female Normal Hiort et al; Am J Med LBD 2622 TTC

 GTC Genet. 63: 218-222, 1996 0369 Prostate Substitute 5 754 Phe

 Leu Somatic mutation Male Normal Takahashi et al; Cancer cancer LBD 2622 TTC

 CTC Research 55: 1621-1624, 1995 0151 PAIS Substitute 5 754 Phe

 Leu Male Ambiguous Hiort et al: Hum Mol LBD 2624 TTC

 TTA Genet 3: 1163-1166 1994 0152 PAIS Substitute 5 * 754 Phe

 Leu normal high * Male Ambiguous Weidemann et al; Clin LBD 2624 TTC

 TTA Endocrinology 45: 733-739, 1996 0370 Prostate Substitute 5 755 Thr

 Ala Somatic mutation Male Normal Takahashi et al; Cancer cancer LBD 2625 ACC

 GCC Research 55: 1621-1624, 1995 0153 PAIS Substitute 5 756 Asn

 Ser Male Ambiguous Hiort et al; Am J Med LBD 2629 AAT

 AGT Genet. 63: 218-222, 1996 0532 MAIS Substitute 5 * 756 Asn

 Ser high Servere oligospermia- Male Normal Giwercman et al. Clin LBD 2629 AAT

 AGT transactivation 38% of Endocrinol 54: 827-834, wt. 2001 0300 Prostate Substitute 5 * 757 Val

 Ala Binds R1881 norm.- Male Normal James et al; 79th US cancer LBD 2632 GTC

 GCC transcriptionally Endo Soc Meeting, inactive-Som mut Abstr P2-484, 1997 0493 Prostate Substitute 5 757 Val

 Ala Somatic mutation Male Normal Marcelli et al; Cancer cancer LBD 2632 GTC

 GCC Research 60: 944-949, 2000 0346 PAIS Substitute 5 * 758 Asn

 Thr normal high * 50% reduction in Yong et al; Mol & Cell LBD 2635 AAC

 ACC transactivation in Endocrinol. 137: 41-50, COS-7 1998 0371 Prostate Substitute 5 759 Ser

 Pro Somatic mutation Male Normal Takahashi et al; Cancer cancer LBD 2637 TCC

 CCC Research 55: 1621-1624, 1995 0154 CAIS Substitute 5 759 Ser

 Phe zero Female Normal DeBellis et al; Mol LBD 2638 TCC

 TTC Endocrinol, 6: 1909-20, 1992 0155 CAIS Substitute 5 762 Leu

 Phe zero Female Normal Brown et al; 74th US LBD 2646 CTC

 TTC Endo Soc Meeting, Abstr 1506, 1992 0156 CAIS Substitute 5 * 762 Leu

 Phe zero Female Normal Bevan et al; J Steroid LBD 2646 CTC

 TTC Biochem Molec. Biol 61: 19-26, 1997 0157 CAIS Substitute 5 763 Tyr

 His Female Normal Quigley et al; Endocrin. LBD 2649 TAC

 CAC Reviews, 16: 271, 1995 0158 PAIS Substitute 5 * 763 Tyr

 Cys 12 normal high * PolyGln tract short Male Ambiguous pos McPhaul et al; J Clin Inv LBD 2650 TAC

 TGC (only 12 repeats) 87: 1413, 1991: Batch&al Arc Dis Ch 68: 453 0159 PAIS Substitute 5 763 Tyr

 Cys low Male Ambiguous Morono et al; Human LBD 2650 TAC

 TGC Mutation 6: 152-162, 1995 0405 PAIS Substitute 5 763 Tyr

 Cys Male Ambiguous Batch et al; Arch LBD 2650 TAC

 TGC Disease Child 68: 453, 1993 0484 PAIS Substitute 5 763 Tyr

 Cys normal high Ahmed et al; J Clin LBD 2650 TAC

 TGC Endocrinol & Metab 85: 658-665, 2000 0485 PAIS Substitute 5 763 Tyr

 Cys normal high Ahmed et al; J Clin LBD 2650 TAC

 TGC Endocrinol & Metab 85: 658-665, 2000 0372 Prostate Substitute 5 763 Tyr

 Cys Somatic mutation Male Normal Takahashi et al; Cancer Cancer LBD 2650 TAC

 TGC Research 55: 1621-1624, 1995 0160 CAIS Substitute 5 * 764 Phe

 Leu low high Female Normal neg Marcelli et al; J clin LBD 2652 TTC

Invest 94: 1642-1650, 1994 0161 CAIS Substitute 5 764 Phe

 Leu zero Female Normal Ris-Stalpers et al: LBD 2652 TTC

 CTC Pediatric Res, 36; 227-234, 1994 0162 CAIS Substitute 5 764 Phe

 Leu low normal Female Normal Pinsky et al; Clin Inv LBD 2654 TTC

 TTG Med, 15: 456, 1992 0163 CAIS Substitute 5 * * 765 Ala

 Thr zero Female Normal Bevan et al; J Steroid LBD 2655 GCC

 ACC Biochem Molec. Biol 61: 19-26, 1997 0164 CAIS Substitute 5 * 765 Ala

 Thr zero Female Normal Merkabi et al; 75th US LBD 2655 GCC

 ACC Endo Soc Meeting Abstr 602, 1993 0165 CAIS Substitute 5 * 765 Ala

 Thr Female Normal Sweet et al; Fertil LBD 2655 GCC

 ACC Sterilty 58: 703, 1992 0166 CAIS Substitute 5 * 765 Ala

 Thr Female Normal Hiort et al; Am J Med LBD 2655 GCC

 ACC Genet. 63: 218-222, 1996 0311 CAIS Substitute 5 * 765 Ala

 Thr 27 Female Normal Ko et al; J Reprod. LBD 2655 GCC

 ACC Med 42: 424-427, 1997 0382 CAIS Substitute 5 * 765 Ala

 Thr Female Normal Giwercman et al; LBD 2655 GCC

 ACC Human Genetics 103: 529-531, 1998 0454 CAIS Substitute 5 * 765 Ala

 Thr Female Normal Ahmed et al; J Clin LBD 2655 GCC

 ACC Endocrinol & Metab 85: 658-665, 2000 0455 CAIS Substitute 5 * 765 Ala

 Thr Female Normal Ahmed et al; J Clin LBD 2655 GCC

 ACC Endocrinol & Metab 85: 658-665, 2000 0456 CAIS Substitute 5 * 765 Ala

 Thr Female Normal Ahmed et al; J Clin LBD 2655 GCC

 ACC Endocrinol & Metab 85: 658-665, 2000 0520 PAIS Substitute 5 765 Ala

 Ser Chavez et al; Clin Genet LBD 2655 GCC

 TCC 59:: 185-188, 2001 0167 CAIS Substitute 5 765 Ala

 Val 20 zero Female Normal Pinsky et al, Clin Inv LBD 2656 GCC

 GTC Med, 15: 456, 1992 0168 CAIS Substitute 5 * 766 Pro

 Ser low high high Female Normal pos Marcelli et al; J Clin LBD 2658 CCT

 TCT Invest 94: 1642-1650. 1994 0457 CAIS Substitute 5 766 Pro

 Ser Female Normal Ahmed et al; J Clin LBD 2658 CCT

 TCT Endocrinol & Metab 85: 658-665, 2000 0543 CAIS Substitute 5 766 Pro

 Ala normal high Female Normal Boehmer et al; J Clin LBD 2658 CCT

 ATG Endocrinol & Metab 86: 4151-4160, 2001 0169 CAIS Deletion 5 766 Pro

 Pro Single nt. deletion Female Normal pos Baldazzi et al; Hum Mol LBD 2660 CCΔT

 CCG causing frameshift & Genet stop in Codon 807 3: 1169-1170, 1994 0388 CAIS Deletion 5 766 Pro

 Pro Single nt. deletion Female Normal Chung et al; Molecules LBD 2660 CCΔT

 CCG causing frameshift & & Cells 8: 741-745, stop in Codon 807 1998 0458 CAIS Deletion 5 766 Pro

 Pro Single nt. deletion Female Normal Ahmed et al; J Clin LBD 2660 CCΔT

 CCG causing frameshift & Endocrinol & Metab 85: stop in Codon 807 658-665, 2000 0459 CAIS Deletion 5 766 Pro

 Pro Single nt. deletion Female Normal Ahmed et al; J Clin LBD 2660 CCΔT

 CCG causing frameshift & Endocrinol & Metab 85: stop in Codon 807 658-665, 2000 0561 CAIS Deletion 5 766 Pro

 Pro Single nt. del framhift Female Normal Guillen et al; An Esp LBD 2660 CCΔT

 CCG & stop in Codon 807 Pediatr 56: 341-352, in 2 unrelated individs 2002 0170 CAIS Substitute 5 767 Asp

 Glu v low Female Normal Lobaccaro et al; Pediatr LBD 2663 GAT

 GAG Res, 33.Abstr 115, 1993 0343 CAIS Substitute 5 767 Asp

 Glu Female Normal Melo et al; 80th US LBD 2663 GAT

 GAG Endo Soc Meeting Abstr P2-44, 1998 0544 PAIS Substitute 5 768 Leu

 Met normal high Female Ambiguous Boehmer et al; J Clin LBD 2664 CTG

 ATG Endocrinol & Metab 86: 4151-4160, 2001 0460 CAIS Substitute 5 768 Leu

 Pro Female Normal Ahmed et al; J Clin LBD 2665 CTG

 CCG Endocrinol & Metab 85: 658-665, 2000 0171 PAIS Substitute 5 771 Asn

 His Female Ambiguous Hiort et al; Hum Mol LBD 2673 AAT

 CAT Genet 3: 1163-1166 1994 0526 PAIS Substitute 5 * 771 Asn

 His high Size & level of Female Ambiguous Zhu et al, 83rd US Endo LBD 2673 AAT

 CAT expression of AR Soc Meeting, Abstr normal P2-34, 2001 0172 CAIS Substitute 5 772 Glu

 Stop zero Female Normal Imasaki et al; Endocrine LBD 2676 GAG

 TAG Journal 42: 643-648 1995 0173 PAIS Substitute 5 772 Glu

 Gly low high Tincello et al; Clinical LBD 2677 GAG

 GGG Endocrinology 46: 497-506, 1997 0174 PAIS Substitute 5 * 772 Glu

 Ala 25 23 normal normal high Male Ambiguous Shkolny et al; J Clin LBD 2677 GAG

 GCG Endocrinol & Metab 84: 805-810, 1999 0336 CAIS Substitute 6 * * 774 Arg

 Cys 26 23 normal normal Female Normal Prior et al; Am J Hum LBD 2682 CGC

 TGC Genet, 51: 143, 1992 0176 CAIS Substitute 6 * * 774 Arg

 Cys 27 19 zero Female Normal pos Prior et al; Am J Hum LBD 2682 CGC

 TGC Genet, 51: 143, 1992 0177 CAIS Substitute 6 * 774 Arg

 Cys zero Female Normal Mebarki et al; 72nd US LBD 2682 CGC

 TGC Endo Soc Meeting, Abstr 791, 1990 0178 CAIS Substitute 6 * 774 Arg

 Cys Female Normal Hiort et al; J Pediatrics LBD 2682 CGC

 TGC 132: 939-943, 1998 0179 CAIS Substitute 6 * 774 Arg

 Cys v low high Female Normal neg Marcelli et al; J Clin LBD 2682 CGC

 TGC Endocrinol & Metab 73: 318, 1991 0180 CAIS Substitute 6 * 774 Arg

 Cys Female Normal Jakubiczka et al; Human LBD 2682 CGC

 TGC Mutation 9: 57-61, 1997 0331 CAIS Substitute 6 * 774 Arg

 Cys Female Normal Komori et al; Arch LBD 2682 CGC

 TGC Gynecol & Obstetrics 261: 95-100, 1998 0175 CAIS Substitute 6 * * 774 Arg

 Cys v low Female Normal Brown et al; Mol LBD 2682 CGC

 TGC Endocrinol, 4: 1759-72, 1990 0355 CAIS Substitute 6 * 774 Arg

 Cys mosaic-de novo Female Normal neg Hiort et al; J Pediatrics LBD 2682 CGC

 TGC mutation 132: 939-943, 1998 0181 CAIS Substitute 6 * * 774 Arg

 His normal high high * mutation found in two Female Normal pos Prior et al; Am J Hum LBD 2683 CGC

 CAC unrelated families Genet, 51: 143, 1992 0182 CAIS Substitute 6 * 774 Arg

 His low normal * Female Normal Batch et al; Hum Mol LBD 2683 CGC

 CAC Genet, 1: 497, 1992 0183 CAIS Substitute 6 * * 774 Arg

 His v low high Female Normal DeBellis et al; Mol LBD 2683 CGC

 CAC Endocrinol, 6: 1909-20, 1992 0184 CAIS Substitute 6 * 774 Arg

 His Female Normal Hiort et al; Am J Med LBD 2683 CGC

 CAC Genet. 63; 218-222, 1996 0461 CAIS Substitute 6 * 774 Arg

 His zero Female Normal Ahmed et al; J Clin LBD 2683 CGC

 CAC Endocrinol & Metab 85: 658-665, 2000 0462 CAIS Substitute 6 * 774 Arg

 His Female Normal Ahmed et al; J Clin LBD 2683 CGC

 CAC Endocrinol & Metab 85: 658-665, 2000 0185 PAIS Substitute 6 * 774 Arg

 His Quicley et al; Endocrin LBD 2683 CGC

 CAC Reviews 16: 271, 1995 0186 CAIS Substitute 6 * 779 Arg

 Trp Female Normal Hiort et al; Hum Mol LBD 2697 CGG

 TGG Genet 3: 1163-1166 1994 0187 CAIS Substitute 6 * * 779 Arg

 Trp Female Normal Morono et al; Human LBD 2697 CGG

 TGG Mutation 6: 152-162, 1995 0188 CAIS Substitute 6 * 779 Arg

 Trp Female Normal Sinnecker et al; Eur J. LBD 2697 CGG

 TGG Pediatr. 156: 7-14, 1997 0463 CAIS Substitute 6 * 779 Arg

 Trp Female Normal Ahmed et al; J Clin LBD 2697 CGG

 TGG Endocrinol & Metab 85: 658-665, 2000 0189 PAIS Substitute 6 * 780 Met

 Ile normal high * Female Ambiguous Bevan et al; Hum Mol LBD 2702 ATG

 ATA Genet, 5: 265-273, 1996 0190 PAIS Substitute 6 780 Met

 Ile 20 23 normal high high 1 family member - Female/ Ambiguous pos Pinsky et al; Clin Inv LBD 2702 ATG

 ATA male. Rest of family - Male Med, 15: 456, 1992 females 0191 PAIS Substitute 6 780 Met

 Ile Brinkmann et al; J LBD 2702 ATG

 ATA Steroid Biochem & Mol Biol 53: 443, 1995 0192 PAIS Substitute 6 780 Met

 Ile A brother to mutation Male Ambiguous pos Rodien et al; J Clin LBD 2702 ATG

 ATA 0305 Endo & Metab 81: 2904-2908, 1996 0305 CAIS Substitute 6 780 Met

 Ile 2 sisters to mutation Female Normal pos Rodien et al; J Clin LBD 2702 ATG

 ATA 0192 Endo & Metab 81: 2904-2908, 1996 0193 CAIS Substitute 6 780 Met

 Ile Female Normal Jakubiczka et al; Human LBD 2702 ATG

 ATA Mutation 9: 57-61, 1997 0464 CAIS Substitute 6 780 Met

 Ile low high Female Normal Ahmed et al; J Clin LBD 2702 ATG

 ATA Endocrinol & Metab 85: 658-665, 2000 0194 Prostate Substitute 6 782 Ser

 Asn Somatic mutation Male Normal Tilley et al; 2: Clinical cancer LBD 2707 AGC

 AAC Cancer Res. 2: 277-285, 1996 0383 CAIS Substitute 6 * 784 Cys

 Tyr zero No transactivation Female Normal Giwercman et al; LBD 2713 TGT

 TAT capacity Human Genetics 103: 529-531, 1998 0195 CAIS Substitute 6 * 786 Arg

 Stop zero Female Normal Pinsky et al; Clin Inv LBD 2718 CGA

 TGA Med, 15: 456, 1992 0557 CAIS Substitute 6 * 786 Arg

 Stop Female Normal Ignaccack et al; J Appl LBD 2718 CGA

 TGA Genet 43: 109-114, 2002 0196 CAIS Substitute 6 * 787 Met

 Val zero Female Normal pos Nakao et al; J Clin LBD 2721 ATG

 GTG Endocrinol Metab, 74: 1152, 1992 0406 MAIS Substitute 6 788 Arg

 Ser 24 23 normal normal high * Gynocomastic and Male Ambiguous pos Lumroso et al 81st. US LBD 2726 AGG

 AGT infertility endo Soc Meetings Abstr. P3-288, 1999 0197 MAIS Substitute 6 * 790 Leu

 Phe normal low * Male Near-normal Tsukada et al; J Clin LBD 2730 CTC

 TTC male Endorinol Metab, 79: 1202, 1994 0198 MAIS Substitute 6 793 Glu

 Asp normal normal Inconsistent increases Male Normal Pinsky et al; Clin Inv LBD 2741 GAG

 GAC in k Med, 15: 456, 1992 0397 Normal Substitute 6 793 Glu

 Asp Homsosexual Male Normal Macke et al; Am J LBD 2741 GAG

 GAC individual Human Genetics 53: 844-852, 1993 0199 CAIS Substitute 6 794 Phe

 Ser Female Normal Hiort et al; Am J Med LBD 2743 TTT

 TCT Genet. 63: 218-222, 1996 0200 CAIS Substitute 6 794 Phe

 Ser Female Normal Jakubiczka et al Human LBD 2743 TTT

 TCT Mutation 9: 57-61, 1997 0201 CAIS Substitute 6 * 796 Trp

 Stop v low Female Normal Marcelli at al; J Clin LBD 2750 TGG

 TGA Invest 85: 1522, 1990 0202 PAIS Substitute 6 * 798 Gln

 Glu normal normal * Female Ambiguous Bevan et al; Hum Mol LBD 2754 CAA

 GAA Genet, 5: 265-273, 1996 0203 PAIS Substitute 6 798 Gln

 Glu normal normal Quigley et al; Endocrine LBD 2754 CAA

 GAA Reviews 16: 271, 1995 0204 PAIS Substitute 6 798 Gln

 Glu Female Ambiguous Hiort et al; Am J Med LBD 2754 CAA

 GAA Genet. 63: 218-222, 1996 0205 Prostate Substitute 6 798 Gln

 Glu Also present in Male Normal Evans et al; Prostate cancer LBD 2754 CAA

 GAA genomic DNA 28: 162-171, 1996 0399 Prostate Substitute 6 798 Gln

 Glu Somatic mutation Male Normal Castagnaro et al; Verh. cancer LBD 2754 CAA

 GAA Stage 4 tumor Dtsch. Ges. Path. 77; 119-123, 1993 0340 MAIS Substitute 6 * 798 Gln

 Glu normal Azospermia Male Normal Hiort et al; J Clin LBD 2754 CAA

 GAA Endocrinol & Metab 85: 2810-2815, 2000 0381 MAIS Substitute 6 * 798 Gln

 Glu normal Azoospermia - Male Normal Wang et al; J Clin LBD 2754 CAA

 GAA defective Endocrinol & Metab 83: transactivation 4303-4309, 1998 0542 CAIS Deletion 6 800 Thr

 Thr Single nt. deletion Female Normal Boehmer et al; J Clin LBD 2762 ACΔC

 ACC causing frameshift & Endocrinol & Metab 86: stop in codon 807 4151-4160, 2001 0521 PAIS Substitute 6 802 Gln

 Arg Chavez et al; Clin Genet LBD 2767 CGG

 CGG 59:: 185-188, 2001 0498 CAIS Substitute 6 803 Glu

 Lys zero zero Female Normal pos Sawai et al. J Hum LBD 2769 GAA

 AAA Genet 45: 342-345, 2000 0206 PAIS Substitute 6 806 Cys

 Tyr Brown et al; Eur J LBD 2779 TGC

 TAC Pediatr 152: (Suppl 2) S62, 1993 0207 CAIS Substitute 6 * 807 Met

 Val low Female Normal Morono et al; Human LBD 2781 ATG

 GTG Mutation 6: 152-162, 1995 0208 CAIS Substitute 6 807 Met

 Arg zero Female Normal Adeyemo et al; Hum LBD 2782 ATG

 AGG Mol Genet, 2: 1809, 1993 0428 PAIS Substitute 6 * 807 Met

 Thr low Treatment with topical Female Ambiguous Ong et al; Lancet 354: LBD 2782 ATG

 ACG DHT restored male 1444-1445, 1999 genital development 0403 PAIS Substitute 6 812 Leu

 Phe Female Normal Yaegashi et al; Tohoku J LBD 2796 CTC

 TTC of Exp Med 187: 263-272, 1999 0209 PAIS Substitute 6 814 Ser

 Asn 20 normal Hormone binding Female Ambiguous Pinsky et al; Clin Inv LBD 2803 AGC

 AAC specificity alterted. Med, 15: 456, 1992 0210 MAIS Substitute 6 814 Ser

 Asn 20 normal Hormone binding Male Normal pos Pinsky et al; Clin Inv LBD 2803 AGC

 AAC specificity altered Med, 15: 456, 1992 0501 CAIS Substitute 7 819 Asp

 Gln Female Normal Choi et al; Arch LBD 2818 GAT

 GGT Gynecol Obstet 263: 201-205, 200 0211 CAIS Substitute 7 * 820 Gly

 Ala normal high * Also Leu 257 Pro, Female Ambiguous neg Tanaka et al; LBD 2821 GGG

 GCG enhances Gynecological Endo. thermolability 12: 75-82, 1998 0212 PAIS Substitute 7 821 Leu

 Val 24 23 normal normal Pinsky et al; Clin Inv LBD 2823 CTG

 GTG Med, 15: 456, 1992 0513 MAIS Substitute 7 * 824 Gln

 Lys Gynocomastia-normal Male Normal pos Giwercman et al; J Clin LBD 2832 CAA

 AAA fertility -related to 514 Endocrinol & Metab 85: abnormal Bmax DHT 2253-2259, 2000 0514 MAIS Substitute 7 * 824 Gln

 Lys Gynocomastia-normal Male Normal pos Giwercman et al; J Clin LBD 2832 CAA

 AAA fertility -related to 513 Endocrinol & Metab 85: abnormal Bmax DHT 2253-2259, 2000 0537 CAIS Substitute 7 827 Phe

 Val Female Normal Chavez et al; J Hum LBD 2841 TTT

 GTT Genet. 46: 560-565, 2001 0522 CAIS Substitute 7 830 Leu

 Val Female Normal Chavez et al; Clin Genet LBD 2850 CTT

 GTT 59:: 185-188, 2001 0213 CAIS Substitute 7 * 831 Arg

 Stop zero Female Normal pos DeBellis et al; Mol LBD 2853 CGA

 TGA Endocrinol, 6: 1909-20, 1992 0214 CAIS Substitute 7 * 831 Arg

 Stop zero Female Normal Tincello et al; J LBD 2853 CGA

 TGA Endocrinol, 132 Suppl, Abstr 87, 1992 0215 CAIS Substitute 7 * 831 Arg

 Stop zero Female Normal Ris-Stalpers et al; 74th LBD 2853 CGA

 TGA Endo Soc Meeting, 1992 0384 CAIS Substitute 7 * 831 Arg

 Stop Female Normal Giwercman et al; LBD 2853 CGA

 TGA Human Genetics 103: 529-531, 1998 0465 CAIS Substitute 7 * 831 Arg

 Stop Female Normal Ahmed et al; J Clin LBD 2853 CGA

 TGA Endocrinol & Metab 85: 658-665, 2000 0500 CAIS Substitute 7 * 831 Arg

 Stop Female Normal Choi et al; Arch LBD 2853 CGA

 TGA Gynecol Obstet 263: 201-205, 2000 0515 CAIS Substitute 7 * 831 Arg

 Stop Harmatoma found in Female Normal Chen et al; Fertilty & LBD 2853 CGA

 TGA pubertal patient Sterility 74: 182-183, 2000 0466 CAIS Substitute 7 * 831 Arg

 Gln Female Normal Ahmed et al; J Clin LBD 2854 CGA

 CAA Endocrinol & Metab 85: 658-665, 2000 0499 CAIS Substitute 7 * 831 Arg

 Gln Female Normal Choi et al; Arch LBD 2854 CGA

 CAA Gynecol Obstet 263: 201-205, 2000 0216 CAIS Substitute 7 * * 831 Arg

 Gln v low Female Normal pos Brown et al; Mol LBD 2854 CGA

 CAA Endocrinol, 4: 1759-72, 1990 0217 CAIS Substitute 7 * 831 Arg

 Gln zero Found in two Female Normal McPhaul et al; J Clin LBD 2854 CGA

 CAA unrelated families Inv, 90: 2097, 1992 0404 CAIS Substitute 7 831 Arg

 Gln zero Female Normal Yaegashi et al; Tohoku J LBD 2854 CGA

 CAA of Exp Med 187: 263-272, 1999 0524 CAIS Substitute 7 831 Arg

 Gln zero Sertoli cell carcinoma Female Normal Ko et al. Int. J. LBD 2854 CGA

 CAA Gynocol. Pathol. 20: 196-199, 2001 0218 CAIS Substitute 7 * 831 Arg

 Leu 21 19 zero Female Normal Shkolny et al; Human LBD 2854 CGA

 CTA Mol Genetics 4: 515-521, 1995 0307 CAIS Substitute 7 * 831 Arg

 Leu 26 16 zero Female Normal Shkolny et al; Human LBD 2854 CGA

 CTA Mol Genetics 4: 515-521, 1995 0219 CAIS Substitute 7 834 Tyr

 Cys zero Female Normal Wilson et al; J Clin LBD 2863 TAC

 TGC Endocrinol Metab, 75: 1474-8, 1992 0392 PAIS Substitute 7 838 Leu

 Leu Hypospadia and Male Ambiguous Nordenskjold et al LBD 2876 CTC

 CTT cryptorchidism - Urological Res, 27: silent mutation 49-55, 1999 0415 PAIS Substitute 7 840 Arg

 Ser Male Ambiguous pos Melo et al; Hum Mutat. LBD 2880 CGT

 AGT 14: 353, 1999 0220 PAIS Substitute 7 * * 840 Arg

 Cys 20 16 normal high norm * Male Ambiguous pos Beitel et al; J Clin Inv, LBD 2880 CGT

 TGT 94: 546-554 1994 0221 PAIS Substitute 7 * * 840 Arg

 Cys low high * Found in two Female McPhaul et al; J Clin LBD 2880 CGT

 TGT unrelated individuals. Inv, 90: 2097, 1992 0222 PAIS Substitute 7 * * 840 Arg

 Cys normal high Sibling of 0308 Female Ambiguous pos Bevan et al; Hum Mol LBD 2880 CGT

 TGT Genet, 5: 265-273, 1996 0308 PAIS Substitute 7 * * 840 Arg

 Cys normal high Sibling of 0222 Male Ambiguous pos Bevan et al; Hum Mol LBD 2880 CGT

 TGT Genet, 5: 265-273, 1996 0387 PAIS Substitute 7 * * 840 Arg

 Cys Transcriptional Georget et al; J Clin LBD 2880 CGT

 TGT activity only at high Endocrinol & Metab 83: conc of androgen 3597-3603, 1998 0385 PAIS Substitute 7 * 840 Arg

 Gly low Reduced Giwercman et al; LBD 2880 CGT

 GGT transactivation Human Genetics 103: 529-531, 1998 0337 PAIS Substitute 7 * * 840 Arg

 His 19 normal high high * Female Ambiguous pos Beitel et al; J Clin Inv, LBD 2881 CGT

 CAT 94: 546-554 1994 0224 PAIS Substitute 7 * * 840 Arg

 His 18 24 normal high high * Female Ambiguous pos Beitel et al; J Clin Inv, LBD 2881 CGT

 CAT 94: 546-554 1994 0225 PAIS Substitute 7 * 840 Arg

 His high Found in two Female Ambiguous pos Hiort et al; J Clin LBD 2881 CGT

 CAT unrelated families in 1 Endocrinol Metab, fam 77: 262-266, 1993 0226 PAIS Substitute 7 * 840 Arg

 His zero McPhaul et al; J Clin LBD 2881 CGT

 CAT Inv, 90: 2097, 1992 0227 PAIS Substitute 7 * 840 Arg

 His normal normal * In same fam. persons Female Ambiguous pos Imasaki et al; Eur J LBD 2881 CGT

 CAT raised as males with Endorinol, 130: ambiguous genitalia 569-574, 1994 0228 PAIS Substitute 7 * 840 Arg

 His low Lumbroso et al; Eur J LBD 2881 CGT

 CAT Endorinol 130: 327, 1994 0229 PAIS Substitute 7 * 840 Arg

 His low Imai et al; Annals of LBD 2881 CGT

 CAT Clinical Biochem, 32: 482-486, 1995 0230 PAIS Substitute 7 * 840 Arg

 His Ghirri & Brown; LBD 2881 CGT

 CAT Pediatr Res 33: Abstr.95, 1993 0231 PAIS Substitute 7 * * 840 Arg

 His low high high Marcelli et al; J Clin LBD 2881 CGT

 CAT Invest 94: 1642-1650, 1994 0232 PAIS Substitute 7 * * 840 Arg

 His normal high * Female Ambiguous pos Weidemann et al; Clin LBD 2881 CGT

 CAT Endocrinology 45: 733-739, 1996 0223 PAIS Substitute 7 * * 840 Arg

 His low high Female Ambiguous De Bellis et al; J Clin LBD 2881 CGT

 CAT Endrcrinol Metab, 78: 513, 1994 0233 PAIS Substitute 7 841 Ile

 Ser Female Ambiguous Hiort et al; Am J Med LBD 2884 ATC

 AGC Genet. 63: 218-222, 1996 0234 CAIS Substitute 7 842 Ile

 Thr Female Normal pos Hiort et al; J Clin LBD 2887 ATT

 ACT Endocrinol Metab, 77: 262-266, 1993 0235 PAIS Substitute 7 * 842 Ile

 Thr low high * Male Ambiguous pos Weidemann et al Clin LBD 2887 ATT

 ACT Endocrinlogy 45: 733-739, 1996 0494 Prostate Substitute 7 846 Arg

 Gly Somatic mutation Male Normal Marcelli et al; Cancer cancer LBD 2898 AGA

 GGA Research 60: 944-949, 2000 0236 CAIS Insertion 7 848 Asn

 Lys zero nt insert causes frame- Female Normal Brinkmann et al; J LBD 2906 AAT

 AAAT shift, stop in Codon Steroid Biochem Mol 879& loss of AA's Biol 53: 443, 1995 0467 CAIS Insertion 7 848 Asn

 Lys zero nt insert causes frame- Female Normal Ahmed et al; Clin LBD 2906 AAT

 AAAT shift. Endocrinol & Metab 85: 658-665, 2000 0237 CAIS Substitute 7 853 Ser

 Stop zero Female Normal Wilson et al; J Clin LBD 2920 TCA

 TGA Endocrinol Metab, 75: 1474-8, 1992 0238 CAIS Substitute 7 853 Ser

 Stop zero Female Normal Jakubiczka et al; Human LBD 2920 TCA

 TGA Mutation 9: 57-61, 1997 0239 PAIS Substitute 7 854 Arg

 Lys low * McPhaul et al; J Clin LBD 2923 AGA

 AAA Inv, 90: 2097, 1992 0240 CAIS Substitute 7 * 855 Arg

 Cys zero Female Normal DeBellis et al; Mol LBD 2925 CGC

 TGC Endocrinol 6: 1909-20, 1992 0241 CAIS Substitute 7 * 855 Arg

 Cys Female Normal Tincello et al; J LBD 2925 CGC

 TGC Endocrinol 132 Suppl, Abstr 87, 1992 0242 CAIS Substitute 7 * 855 Arg

 Cys zero Female Normal McPhaul et al; J Clin LBD 2925 CGC

 TGC Inv, 90: 2097, 1992 0243 CAIS Substitute 7 * 855 Arg

 Cys Female Normal Loboccaro et al; Pediat LBD 2925 CGC

 TGC Res 33: Abstr 115, 1993 0244 CAIS Substitute 7 * * 855 Arg

 Cys low Female Normal pos Morono et al; Human LBD 2925 CGC

 TGC Mutation 6: 152-162. 1995 0245 CAIS Substitute 7 * 855 Arg

 Cys zero Female Normal Sultan et al; J Steroid LBD 2925 CGC

 TGC Biochem & Mol Biol: 40 519, 1993 0246 CAIS Substitute 7 * 855 Arg

 Cys Female Normal Brinkmann et al; J LBD 2925 CGC

 TGC Steroid Biochem & Mol Biol 53: 443, 1995 0247 CAIS Substitute 7 * 855 Arg

 Cys Female Normal Hiort et al; Am J Med LBD 2925 CGC

 TGC Genet. 63: 218-222, 1996 0248 CAIS Substitute 7 * 855 Arg

 Cys v low high Female Normal pos Malmgren et al; Clin LBD 2925 CGC

 TGC Genet. 50: 202-205, 1996 0320 CAIS Substitute 7 * 855 Arg

 Cys Female Normal Komori et al: J LBD 2925 CGC

 TGC Obstetrics & Gynocol. Res. 23: 277-81, 1997 0468 CAIS Substitute 7 * 855 Arg

 Cys zero Female Normal Ahmed et al; J Clin LBD 2925 CGC

 TGC Endocrinol & Metab 85: 658-665, 2000 0469 CAIS Substitute 7 * 855 Arg

 Cys normal high Female Normal Ahmed et al; J Clin LBD 2925 CGC

 TGC Endocrinol & Metab 85: 658-665, 2000 0527 CAIS Substitute 7 * * 855 Arg

 Cys v low high Female Normal Elhaji et al. 83rd US LBD 2925 CGC

 TGC Endo Soc Meeting, Abstr P2-37, 2001 0528 PAIS Substitute 7 * * 855 Arg

 His normal high * Male Ambiguous Elhaji et al. 83rd US LBD 2925 CGC

 CAC Endo Soc Meeting, Abstr P2-37, 2001 0251 PAIS Substitute 7 * 855 Arg

 His normal high Chang et al; 73rd Endo LBD 2926 CGC

 CAC Soc Meeting, Abstr 28, 1991 0252 PAIS Substitute 7 * * 855 Arg

 His normal high * Servere hypospadia Male Ambiguous pos Batch et al; Hum Mol LBD 2926 CGC

 CAC Genet, 1: 497, 1992 0253 PAIS Substitute 7 * 855 Arg

 His Male Ambiguous Hiort et al; Am J Med LBD 2926 CGC

 CAC Genet. 63: 218-222. 1996 0254 PAIS Substitute 7 * * 855 Arg

 His zero Female Ambiguous pos Weidemann et al; Clin LBD 2926 CGC

 CAC Endocrinology 45: 733-739, 1996 0255 PAIS Substitute 7 * * 855 Arg

 His low high norm Female Ambiguous Marcelli et al; J Clin LBD 2926 CGC

 CAC Invest, 94: 1642-1650, 1994 0301 PAIS Substitute 7 * 855 Arg

 His 14 Brother of 0302 Male Ambiguous pos Boehmer et al; Am J LBD 2926 CGC

 CAC somatic & germ-line Hum Genetics 60: muts. in mother 1003-6, 1997 0250 PAIS Substitute 7 * 855 Arg

 His zero Female Ambiguous Weidemann et al; Clin LBD 2926 CGC

 CAC Endorinology 45: 733-739, 1996 0302 PAIS Substitute 7 * * 855 Arg

 His 14 Sister of 0301. Female Ambiguous pos Boehmer et al; Am J LBD 2926 CGC

 CAC somatic & germ-line Hum Genetics 60: muts. in mother 1003-6, 1997 0249 CAIS Substitute 7 * 855 Arg

 His low Female Normal McPhaul et al; J Clin LBD 2926 CGC

 CAC Invest. 90: 2097, 1992 0344 PAIS Substitute 7 * 855 Arg

 His Melo et al; 80th US LBD 2926 CGC

 CAC Endo Soc Meetings Abstr P2-44, 1998 0470 CAIS Substitute 7 856 Phe

 Leu Female Normal Ahmed et al; J Clin LBD 2930 TTC

 TTG Endocrinol & Metab 85: 658-665, 2000 0356 CAIS Substitute 7 857 Tyr

 Stop de novo mutation Female Normal neg Hiort et al; J Pediatrics LBD TAC

132: 939-943, 1998 0256 CAIS Substitute 7 863 Leu

 Arg Female Normal Brown et al; Eur J LBD 2950 CTG

 CGG Pediatr 152: (Suppl 2) S62, 1993 0257 CAIS Substitute 7 * 864 Asp

 Asn low Transactivation Female Normal Bevan et al; J Steroid LBD 2952 GAC

 AAC activity increases with Biochem Molec. Biol horm. concentration 61: 19-26, 1997 0471 CAIS Substitute 7 864 Asp

 Asn Female Normal Ahmed et al; J Clin LBD 2952 GAC

 AAC Endocrinol & Metab 85: 658-665, 2000 0258 CAIS Substitute 7 * 864 Asp

 Gly zero Female Normal DeBellis et al; Mol LBD 2953 GAC

 GGC Endocrinol, 6: 1909-20, 1992 0472 CAIS Substitute 7 864 Asp

 Gly zero Female Normal Ahmed et al; J Clin LBD 2953 GAC

 GGC Endocrinol & Metab 85: 658-665, 2000 0486 CAIS Substitute 7 865 Ser

 Pro Female Normal Ahmed et al; J Clin LBD 2955 TCC

 CCC Endocrinol & Metab 85: 658-665, 2000 0560 CAIS Substitute 7 865 Ser

 Pro de novo mut. also Female Normal pos Mongan et al; J Clin LBD 2955 TCC

 CCC Phe868Leu mut-no endocrinol Metab 87: effect horm binding 1057-1061, 2002 0259 PAIS Substitute 7 866 Val

 Leu 21 normal high Male Ambiguous pos Saunders et al; Clin LBD 2958 GTG

 TTG Endocrinol 37: 214, 1992 0345 PAIS Substitute 7 866 Val

 Leu 25 normal high Male Ambiguous Saunders et al; Clin LBD 2958 GTG

 TTG Endocrinol, 37: 214, 1992 0260 PAIS Substitute 7 * 866 Val

 Leu normal high Male Ambiguous pos Kazemi-Esfarjani et al; LBD 2958 GTG

 TTG Mol Endocrinol, 7: 37-46, 1993 0261 PAIS Substitute 7 866 Val

 Leu high Male Ambiguous pos Hiort et al; J Clin LBD 2958 GTG

 TTG Endocrinol Metab, 77: 262-266, 1993 0262 PAIS Substitute 7 * 866 Val

 Leu zero Merkabi et al; 75th US LBD 2958 GTG

Endo Soc Meeting Abstr 602, 1993 0263 CAIS Substitute 7 * * 866 Val

 Met 20 16 normal high Female Normal Kazemi-Esfarjani et al; LBD 2958 GTG

 ATG Mol Endocrinol, 7: 37-46, 1993 0264 CAIS Substitute 7 * * 866 Val

 Met normal high Female Normal Weidemann et al; Clin LBD 2958 GTG

 ATG Endocrinology 45: 733-739, 1996 0265 CAIS Substitute 7 * 866 Val

 Met normal high * Female Normal Lubahn et al; Proc Natl LBD 2958 GTG

 ATG Acad Sci. 86: 9534, 1989 0266 PAIS Substitute 7 * 866 Val

 Met * McPhaul et al; J Clin LBD 2958 GTG

 ATG Inv, 90: 2097, 1992 0267 PAIS Substitute 7 * 866 Val

 Met high * de novo mutation- Female Ambiguous neg Hiort et al; J Pediatrics LBD 2958 GTG

 ATG mosaic 2 functionally 132: 939-943, 1998 diff AR's 0373 Prostate Substitute 7 * 866 Val

 Met Somatic mutation Male Normal Takahashi et al; Cancer cancer LBD 2958 GTG

 ATG Research 55: 1621-1624, 1995 0473 CAIS Substitute 7 * 866 Val

 Met Female Normal Ahmed et al; J Clin LBD 2958 GTG

 ATG Endocrinol & Metab 85: 658-665, 2000 0474 CAIS Substitute 7 * 866 Val

 Met zero Female Normal Ahmed et al; J Clin LBD 2958 GTG

 ATG Endocrinol & Metab 85: 658-665, 2000 0475 CAIS Substitute 7 * 866 Val

 Met zero Female Normal Ahmed et al; J Clin LBD 2958 GTG

 ATG Endocrinol & Metab 85: 658-665, 2000 0268 CAIS Substitute 7 866 Val

 Glu Female Normal McPhaul et al; J Clin LBD 2959 GTG

 GAG Inv, 90: 2097, 1992 0269 PAIS Substitute 8 * 869 Ile

 Met normal high * Hypospadia Male Ambiguous pos Bevan et al; Hum mol LBD 2969 ATT

 ATG Genet, 5: 265-273, 1996 0270 PAIS Substitute 8 * 870 Ala

 Val Found in two Male Ambiguous Hiort et al; Eur J Pediatr, LBD 2971 GCG

 GTG unrelated families 153: 317, 1994 0315 PAIS Substitute 8 870 Ala

 Gly Servere hypospadias Male Ambiguous Albers et al; J of LBD 2971 GCG

 GGG Pediatrics 131: 388-392, 1997 0271 PAIS Substitute 8 * 870 Ala

 Gly de novo mutation Female Ambiguous neg Hiort et al; J Pediatrics LBD 2971 GCG

 GGG 132: 939-943, 1998 0562 MAIS Substitute 8 * 870 Ala

 Gly bilateral gynecomastia Male Normal Zenteno et al; Horm Res LBD 2971 GCG

 GGG 57: 90-93, 2002 0272 MAIS Substitute 8 * 871 Arg

 Gly 26 24 normal normal norm Male Normal Shkolny et al; J Clin LBD 2973 AGA

 GGA Endocrinol & Metab 84: 805-810, 1999 0273 Prostate Substitute 8 874 His

 Tyr Som mut-stimulated Male Normal Taplin et al; New cancer LBD 2982 CAT

 TAT by progesterone & England J Med 332: oestrogen 1393-1398, 1995 0274 Prostate Substitute 8 874 His

 Tyr Somatic mutation Male Normal Tan et al; J of Urology cancer LBD 2982 CAT

 TAT 155: 340A, 1996 0538 CAIS Substitute 8 874 His

 Arg zero Female Normal Chavez et al; J Hum LBD 2983 CAT

 CGT Genet. 46: 560-565, 2001 0275 LNCaP Substitute 8 877 Thr

 Ala Altered binding Male Normal Veldscholte et al; mutation LBD 2991 ACT

 GCT specificity-somatic Biochem Biophys Res mutation Comm, 172: 534, 1990 0276 Prostate Substitute 8 877 Thr

 Ala Somatic mutation ⅛ Male Normal Suzuki et al; J Steroid cancer LBD 2991 ACT

 GCT endocrine resistant Biochem Molec Biol therapy cases 46: 759, 1993 0277 Prostate Substitute 8 877 Thr

 Ala 6 out of 24 patients Male Normal Gaddipati et al; Cancer cancer LBD 2991 ACT

 GCT screened-somatic Res, mutation 54: 2861-2864, 1994 0278 Prostate Substitute 8 877 Thr

 Ala 3 out of 22 cases in Male Normal Suzuki et al; Prostate cancer LBD 2991 ACT

 GCT metastatic tissue- 29: 153-158, 1996 somatic mutation 0279 Prostate Substitute 8 877 Thr

 Ala Somatic mutation in Male Normal Kleinerman et al; J of cancer LBD 2991 ACT

 GCT bone metasteses of Urology 155: 624A, Prostate cancer 1996 0432 Prostate Substitute 8 877 Thr

 Ala Som mut found in 5 Male Normal Taplin et al; Cancer cancer LBD 2991 ACT

 GCT of 16 patients treated Research 59: 2511-2515 with flutamide 1999 0280 Prostate Substitute 8 * 877 Thr

 Ser Som mut. in 86% of Male Normal Taplin et al: New cancer LBD 2992 ACT

 AGT isolates.Stimulated by England J Med 332: estrogen & progest 1393-1398, 1995 0539 PAIS Substitute 8 879 Asp

 Tyr normal Male Ambiguous Chavez et al; J Hum LBD 2997 GAC

 TAC Genet. 46: 560-565, 2001 0553 Prostate Substitute 8 879 Asp

 Gly Treated with Male Normal Taplin et al; 37th cancer LBD 2998 GAC

 GCC bicalumatide-somatic meeting ASCO 20: mutatation Abstr 1738, 2001 0281 CAIS Substitute 8 881 Leu

 Val Somatic instabilty in Female Normal pos Davies et al; Clinical LBD 3003 CTA

 GTA polyglutamine tract Endocrinology 43: 69-77, 1995 0282 CAIS Substitute 8 883 Lys

 Stop zero Female Normal pos Trifiro et al; Am J Med LBD 3009 AAG

 TAG Genet, 40: 493, 1991 0283 MAIS Substitute 8 * 886 Met

 Val 23 23 normal normal norm Oligospermia-50% Male Normal Yong et al; 46th Am Soc LBD 3018 ATG

 GTG red. in transactivation Hum Genetics meetings Abstr 217, A43, 1996 0309 MAIS Substitute 8 * 886 Met

 Val 21 24 normal normal norm Oligospermia-50% Male Normal Yong et al; 46th Am Soc LBD 3018 ATG

 GTG red. in transactivation Hum Genetics meetings Abstr 217, A43, 1996 0533 PAIS Substitute/ 8 * 888 Ser

 Ser 21 24 v low normal silent mut.-part exon Male Ambiguous Hellwinkel et al. J Clin Splice LBD 3026 AGC

 AGT 8 + part of 3′ untransl Endocrinol & Metab 86: also small amt. wt AR 2569-2575, 2001 0540 PAIS Substitute/ 8 * 888 Ser

 Ser normal Male Ambiguous Chavez et al; J Hum Splice LBD 3026 AGC

 AGT Genet. 46: 560-565, 2001 0476 CAIS Substitute 8 * 889 Val

 Met low normal Female Normal Ahmed et al; J Clin LBD 3027 GTG

 ATG Endocrinol & Metab 85: 658-665, 2000 0284 CAIS Substitute 8 * 889 Val

 Met zero Female Normal Pinsky et al; Clin Inv LBD 3027 GTG

 ATG Med, 15: 456, 1992 0285 PAIS Substitute 8 * * 889 Val

 Met low normal Female Normal De Bellis et al; J Clin LBD 3027 GTG

 ATG Endocrinol Metab, 78: 513, 1994 0321 PAIS Substitute 8 * 889 Val

 Met Female Normal Essawi et al; Disease LBD 3027 GTG

 ATG Markers 13: 99-105, 1997 0433 Prostate Substitute 8 * 890 Asp

 Asn Mutation also found Male Normal Taplin et al; Cancer cancer LBD 3030 GAC

 AAC in peripheral blood Research 59: 2511-2515, 1999 0389 CAIS Substitute 8 * 892 Pro

 Leu 26 low high Reduced Female Normal neg Peters et al; Mol & LBD 3036 CCG

 TCG transactivation Cellular Endocrinol. 148: 47-53, 1999 0375 CAIS Substitute 8 892 Pro

 Leu Mutation found in two Female Normal pos Knoke et al; Human LBD 3037 CCG

 CTG siblings Mutation 12: 220, 1998 0413 CAIS Substitute 8 892 Pro

 Leu Female Normal Kanayama et al; Int J LBD 3037 CCG

 CTG Urology 6: 327-330, 1999 0386 CAIS Substitute 8 * 895 Met

 Thr low Reduced Female Normal Giwercman et al; LBD 3046 ATG

 ACG transactivation Human Genetics 103: 529-531, 1998 0286 CAIS Substitute 8 898 Ile

 Thr de novo mutation Female Normal neg Hiort et al; J Pediatrics LBD 3055 ATC

 ACC 132: 939-943, 1998 0287 Prostate Substitute 8 902 Gln

 Arg Somatic mutation in Male Normal Taplin et al; New cancer LBD 3066 CAA

 CGA 37% of isolates in England J Med 332: initial cloning 1393-1398, 1995 0288 PAIS Substitute 8 903 Val

 Met low Qualitative binding McPhaul et al; J Clin LBD 3069 GTG

 ATG abnormality Inv, 90: 2097, 1992 0289 CAIS Substitute 8 904 Pro

 Ser 27 23 normal high Female Normal Pinsky et al; Clin Inv LBD 3072 CCC

 TCC Med, 15: 456, 1992 0290 CAIS Substitute 8 904 Pro

 His zero Female Normal McPhaul et al; J Clin LBD 3073 CCC

 CAC Inv, 90: 2097, 1992 0291 CAIS Substitute 8 * 907 Leu

 Phe low normal Decreased Female Normal Bevan et al; J Steroid LBD 3081 CTT

 TTT transactivation activity Biochem Molec. Biol compared to normal 61: 19-26, 1997 0292 PAIS Substitute 8 * 909 Gly

 Arg low low Also silent G to A Female Ambiguous pos Choong et al; J Clin LBD 3087 GGG

 AGG mutation in codon 211 Endocrinol Metab, 81: 236-243, 1996 0374 Prostate Substitute 8 909 Gly

 Glu Somatic mutation Male Normal Takahashi et al; Cancer cancer LBD 3088 GGG

 GAG Research 55: 1621-1624, 1995 0327 Prostate Substitute 8 910 Lys

 Arg Somatic mutation Male Normal Watanabe et al; Jpn J cancer LBD 3091 AAA

 AGA Clin Oncol 27: 389-393, 1997 0430 PAIS Substitute 8 911 Val

 Leu 19 Servere Male Ambiguous Knoke et al; Andrologia LBD 3093 GTC

 CTC oligozoospermia 31: 199-201, 1999 0293 PAIS Substitute 8 913 Pro

 Ser Ghirri and Brown; Paed LBD 3099 CCC

 TCC Res, 33(5) Suppl, Abstr 95, 1993 0318 CAIS Substitute 8 * 916 Phe

 Leu low high * Female Normal Radnayr et al; J of LBD 3110 TTC

 TTG Urology 158: 1553-1556, 1997 0477 CAIS Substitute 8 917 His

 Arg Female Normal Ahmed et al; J Clin LBD 3112 CAC

 CGC Endocrinol & Metab 85: 658-665, 2000 0303 Prostate Substitute 8 * 919 Gln

 Arg Somatic mutation Male Normal Nazareth et al; 79th US cancer LBD 3118 CAG

 CGG Endo Soc Meetings Abstr. P2-489, 1997 0294 CAIS Splice exon1

24 23 Insertion at +3 Female Normal Trifiro et al; Eur J Hum intron1 gta

 gtta position of donor Genetics 5: 50-58, 1997 splice site 0304 CAIS Splice exon2

Substitution at +1 Female Normal neg Hellwinkel et al; J intron2 ctg

 cta pos of donor splice Steroid Biochem & Mol site-lacks exon 2 Biol 68: 1-9, 1999 0479 CAIS Splice exon2

zero Female Normal Ahmed et al; J Clin intron2

Endocrinol & Metab 85: 658-665, 2000 0480 CAIS Splice exon2

Female Normal Ahmed et al; J Clin intron2

Endocrinol & Metab 85: 658-665, 2000 0295 CAIS Splice exon3

Substitution at +1 Female Normal Evans et al; J Endocrinol intron3 ggt

 gat position of donor 129 Suppl, Abstr 65, splice site 1991 0478 CAIS Splice exon3

normal normal Substitution at +1 Female Normal Ahmed et al; J Clin intron3 ggt

 gat position of donor Endocrinol & Metab 85: splice site 658-665, 2000 0296 CAIS Splice exon4

zero +1 pos of donor site. Female Normal Ris-Stalpers et al; Proc intron4 ggt

 gtt Splice site activated & Natl AcadSci del of aa's 683-723 87: 7866-70, 1990 0297 CAIS Splice exon6

21 zero Substitution at +3 Female Normal pos Pinsky et al; Eur J Hum intron6 gta

 tta position of donor Genetics 5: 50-58, 1997 splice site 0503 PAIS Splice exon6

low normal Subst. at +5 position Female Ambiguous Sammarco et al; J Clin intron6 taa

 tat of donor splice site, Endocrinol & Metab 85: stop at +79 bases 3256-3261, 2000 0541 CAIS Splice exon6 aag

 aac zero Sust. at +6 position Female Normal Chavez et al; J Hum intron6

of donor splice site. Genet 46: 560-565, 2001 0298 CAIS Splice exon7

zero Subst. at +1 pos of Female Normal pos Lim et al; Mol & Cell intron7 tgt

 tat donor splice, -exon7, Endocrinology 131: stop +10 aa exon 8 205-210, 1997 0502 CAIS Splice exon7

Sustitution at +1 Female Normal Choi et al; Arch intron7 tgt

 tat position of donor Gynecol Obstet 263: splice site 201-205, 200 0299 PAIS Splice intron2/

Subst. at −11 pos of Male Normal Bruggenwirth et al; exon3 gtt

 gat acceptor site. 2 transc; Am J Hum Genet 61: 1, -exon3, 1, +69 nt. 1067-1077, 1997 0317 Breast Splice

-exon 3: higher Female Normal Zhu et al; Intl J of Cancer

express. of mut. var in Cancer 72: 574-580, 7/31breast cancer 1997 0351 CAIS Substitute intron2

Female Normal Hiort et al; J Pediatrics gt

 at 132: 939-943, 1998 0088 PAIS Deletion intron2

normal normal 6 kb del at −18 pos of Male Ambiguous pos Ris-Stalpers et al; Am J

acceptor site 2 transcr: Hum Genet 54: 609, 1 wt, 1 minus exon 3 1994 0312 Prostate Substitute 5′

+2 pos from Male Normal Crociotto et al; J of Cancer UTR agc

 atc transcription initiation Urology 158: site AR-TIS II 1599-1601, 1997 0313 Prostate Substitute 5′

+214 pos from Male Normal pos Crociotto et al; J of Cancer UTR gcc

 gac transcription initiation Urology 158: site AR-TIS II 1599-1601, 1997 0323 Prostate 3′

Som mut. polymorph Male Normal Paz et al; European Cancer UTR

seq 2820-36 dwnstrm Urology 31: 209-215, to transl. init. site 1997

Other advantages and characteristics will become apparent from the examples below pertaining to:

-   -   Example 1: Inhibition of the protein PML-RARα associated with         acute promyelocytic leukemia (APL).     -   Example 2: Inhibition of the tumoral angiogenesis induced by         VEGF.     -   Example 3: Inhibition of the hypoxic response induced by HIF1α.     -   Example 4: Inhibition of the wild or mutant forms of the         androgen receptors in prostate carcinoma cells.     -   Example 5: Inhibition of the wild or mutant forms of the protein         p53.     -   Example 6: Inhibition of the viral protein E6.     -   Example 7: Use of DNA/RNA hybrids to inhibit the expression of         various proteins.     -   Example 8: In vivo administration of siRNA via different routes.

EXAMPLE 1 Inhibition of the Protein PML-RARα Associated with Acute Promyelocytic Leukemia (APL)

I—Introduction

Acute promyelocytic leukemia (APL) is due to the translocation t(15;17) on chromosome 15. In patients afflicted with APL, the receptor of retinoic acid (RARα) is fused to the protein PML (promyelocytic leukemia protein) thereby generating the fusion protein PML-RARα. Five fusion proteins bringing RARα into play have been identified to date. All of these leukemia types implicate the RARα receptor and are clinically similar, which suggests that the rupture of the transduction pathway of retinoic acid is crucial in the pathogenesis of APL leukemia.

The fusion protein PML-RARα retained the binding domains of the DNA and retinoic acid of the RARα. It has been shown that the fusion protein PML-RARα represses the expression of the target genes of retinoic acid and thereby also blocks the differentiation of the promyelocytic cells. Only the administration of pharmacological doses of retinoic acid remove transcriptional repression exerted by PML-RARα and restore cellular differentiation. Moreover, the protein portion PML of the fusion protein could also intervene in the mechanism of the blocking of the transduction pathway by retinoic acid. To the extent that PML functions as a growth inhibitor and an apoptotic agent and that it is required for the expression of certain genes induced by retinoic acid, the dominant negative effect of PML-RARα on PML could allow cells to acquire a growth capacity, a resistance to apoptosis and a termination of differentiation.

Cellular biology studies of PML have shown that this protein possesses a particular localization in the nucleus, in structures called nuclear bodies. It appears that these structures are in direct relation with the anti-oncogene role of PML. In malignant APL cells, the protein PML-RARα induces, by heterodimerization with PML, the delocalization of PML from the nuclear bodies to the micropunctuated structures that could correspond to PML-RARα anchorage sites on the chromatin. This delocalization could block the pro-apoptotic function of PML and its role in myeloid differentiation. Multiple research teams have shown that combined treatment with retinoic acid and AS₂O₃ on cell lines that express the fusion protein PML-RARα enable the degradation of the fusion proteins at the same time as a relocalization of PML on the nuclear bodies. This reorganization of the nuclear bodies restores the functions of PML and contributes to the restoration of differentiation.

Finally, the chimera protein PML-RARα would thus have a double dominant negative effect on RARα and on PML enabling the cells to escape from apoptosis and blocking the differentiation of the thereby transformed promyelocytes.

More than 98% of the patients suffering from APL leukemia present the translocation t(15;17) (q22;q21) which leads to the formation of fused genes PML-RARAα and RARα-PML. There exist two subtypes of fusion proteins PML-RARα: the S (short) fusions and the L (long) fusions). The long form of the fusion protein PML-RARα corresponding to a protein of 955 amino acids representing the predominantly expressed form, and thus was taken as model in this study (Tables 2, 3 and 5). This protein comprises amino acids 1 to 552 of the protein PML fused with amino acids 59 to 462 of the α receptor of retinoic acid (RARα).

II—Preparation and Administration of the Oligonucleotides

Complementary RNA oligonucleotides corresponding to the sequence of the junction of the gene of the fusion protein, i.e., 10 nucleotides of the PML gene and 10 nucleotides of the RARα gene, were synthesized with addition of two deoxythymidines at 3′ (FIG. 1). These oligonucleotides were hybridized and the production of the double-strand oligonucleotide was verified on acrylamide gel.

The sequences of the PML-RAR and control siRNAs used (5′-3′) are presented below:

Control:

FW: [CAUGUCAUGUGUCACAUCUC]RNA[TT]DNA (SEQ ID NO.3) REV: [GAGAUGUGACACAUGACAUG]RNA[TT]DNA (SEQ ID NO.4) PR: Sense: [GGGGAGGCAGCCAUUGAGAC]RNA[TT]DNA (SEQ ID NO.5) Antisense: [GUCUCAAUGGCUGCCUCCCC]RNA[TT]DNA (SEQ ID NO.6) III—Results

NIH3T3 fibroblasts were cotransfected with lipofectamine by an expression vector of the protein PML-RARα (100 ng) and by 500 ng of control siRNA (C) or siRNA directed against PML-RARα (PR). 48 h after transfection, a Western blot (FIG. 1B) was performed on the total cell extracts using an antibody which recognized the protein RARα, whole or in fusion protein form.

FIG. 1B shows that the transfection of siRNA PR very strongly inhibits the expression of fusion protein PML-RARα compared to the cells transfected with the control siRNA (C) without modifying the expression of the protein RARα.

EXAMPLE 2 Inhibition of Tumoral Angiogenesis by VEGF

I—Introduction

VEGF (vascular endothelial growth factor) is one of the most powerful angiogenic factors identified. These factors are overexpressed in numerous situations of pathological hypervascularization and notably in tumoral development. The inhibition of this angiogenesis enables blocking of tumor growth. The method has the goal of inhibiting tumoral angiogenesis by blocking the expression of one of these angiogenic factors, and as seen in this example, that of VEGF by the tumor cells.

II—Preparation and Administration of the Oligonucleotides

Two RNA oligonucleotides, complementary of a region of the coding sequence of human VEGF, conserved in the rat and the mice, were synthesized. Two deoxynucleotides (TT) were added at 3′:

Sequence of the RNAi VEGF: 5′[AUGUGAAUGCAGACCAAAGAA]RNA-TT[DNA] (SEQ ID NO.7) 5′[UUCUUUGGUCUGCAUUCACAU]RNA-TT[DNA] (SEQ ID NO.8) Sequence of the control RNAi: 5′[CAUGUCAUGUGUCACAUCUC]RNA-TT[DNA] (SEQ ID NO.9) 5′[GAGAUGUGACACAUGACAUg]RNA-TT[DNA] (SEQ ID NO.10)

These oligonucleotides or the control oligonucleotides, whose sequence presents no homology with the sequences stored in the data banks, were hybridized and transfected using the Polyfect kit (Qiagen) in the cells of a rat fibrosarcoma (cJ4) and in human cells of the prostate carcinoma LNCaP.

III—Results

48 h after transfection, an indirect immunofluorescence was performed to detect the expression of the protein in the cells. FIG. 2A shows a massive inhibition of the expression of VEGF.

In order to quantify this effect, quantitative determination of the VEGF in the transfected CJ4 cells in parallel with the control RNAi or with the RNAi VEGF was performed with ELISA (quantikine, R&D). The cells were incubated for 48 h prior to the quantitative determination in a medium containing 1% serum. The determination was performed 4 days and 6 days after transfection. Under these conditions, FIG. 2B shows an inhibition of the secretion of VEGF of 85% at 4 days and of 75% at 6 days and of 60% at 13 days in the cells transfected with the RNAi VEGF compared to the cells transfected with the control RNAi (FIG. 2B).

The effect of the inhibition of VEGF on the tumor cells was tested in vivo: 3 days after transfection, three groups of 4 female nude mice aged 4 weeks were injected subcutaneously at the rate of one million cells per mouse: the first group was injected with nontransfected cells, the second group was injected with cells transfected by the control RNAi, the third group was injected with cells transfected with RNAi VEGF. No selection of the transfected cells was performed before the injection.

Tumor growth was monitored by measuring the volume of the tumors at regular intervals (FIG. 2C).

FIGS. 2C and 2D do not show any significant difference between the sizes of the tumors in groups A and B. A very large reduction in the tumor volume was seen in group C. The appearance of the tumors, much whiter in group C (FIG. 2D), manifested a pronounced decrease in the tumoral vascularization. After sacrifice of the animals on day 12 after the injection, the tumors were dissected, fixed and immunodetection of VEGF was performed on sections of these tumors. There was seen a very strong reduction in the expression of VEGF in the tumors of group C compared to that of group B (FIG. 2E).

In another experiment, tumors were induced in male nude mice by injection of prostate carcinoma cells LNCaP. 40 days after injection, the volume of the tumors having reached 1 to 1.5 cm³, the mice were divided into two groups. The first group (4 mice) received an intravenous injection in the tail vein of 2 micrograms of control siRNA in 100 μl of PBS. The second group received an equivalent dose of siRNA VEGF under the same conditions. It was observed that the siRNA VEGF but not the control siRNA induced a transitory suspension in tumor growth (FIG. 4D).

EXAMPLE 3 Inhibition of the Hypoxic Reaction

I—Introduction

Certain tumors are capable of developing under strongly anoxic conditions. This is seen very frequently in tumors of regions that are very poorly vascularized. This weak sensitivity to hypoxia has two consequences: on the one hand, an antiangiogenic treatment has little chance of being effective on these tumors or these tumor subpopulations. On the other end, this weak vascularization makes it difficult to deliver the therapeutic molecules. The transcription factor Hif1α regulates the activity of more than 100 genes enabling the hypoxic response. The inhibition of this transcription factor in hypoxic tumors has the goal of blocking their growth.

II—Preparation of the oligonucleotides

RNAi Hiflα 5′[CAUGUGACCAUGAGGAAAUGA]RNA-TT[DNA] (SEQ ID NO.11) 5′[UCAUUUCCUCAUGGUCACAUG]RNA-TT[DNA] (SEQ ID NO.12) Control RNAi 5′[GAUAGCAAUGACGAAUGCGUA]RNA-TT[DNA] (SEQ ID NO.13) 5′[UACGCAUUCGUCAUUGCUAUC]RNA-TT[DNA] (SEQ ID NO.14) III—Results

The VEGF promoter contains a response element to the transcription factor Hif1α. In order to test in vitro the effect of an RNAi directed against Hif1α, we transfected cJ4 cells with a reporter vector VEGF-luciferase, alone or in combination with an RNAi Hif1α or control.

24 h after transfection, the cells were incubated for 18 h in medium without serum, with the addition in some cases of cobalt chloride 100 μM in order to produce hypoxic conditions; the luciferase activity was then measured.

FIG. 3 shows that a complete inhibition of the induction of the response of the promoter VEGF to hypoxia was observed when the cells were transfected with RNAi Hif1α but not with the control RNAi.

EXAMPLE 4 Inhibition of the Wild or Mutant Forms of the Androgen Receptors in Prostate Carcinomas

I—introduction

The prostate carcinomas are the second cause of cancer mortality for men in the industrialized countries. They are the cause of more than 9500 deaths per year in France. The prostatic epithelial cells are dependent on androgens for their growth. Prostatic carcinomas are initially androgen dependent. Chemical castration thus initially can block the growth of the carcinoma. However, in all cases, these carcinomas become androgen independent and their prognosis becomes very negative. This androgen independence—depending on the individuals—is often due to a mutation of the receptor (conferring on it, for example, a response to estrogens or to glucocorticoids) or to an amplification of the receptor.

II—Preparation of the Oligonucleotides

Two RNA oligonucleotides complementary to a region of the coding sequence of the nonmutated human androgen receptor (AR) were synthesized. Two deoxynucleotides (TT) were added at 3′. In other experiments, siRNAs named LNCaP and specifically recognizing the mutation of the androgen receptor (T877A) in the cells of human prostate carcinoma LNCaP, were used.

AR: 5′[GACUCAGCUGCCCCAUCCACG]RNA-TT[DNA] (SEQ ID NO.15) 5′[CGUGGAUGGGGCAGCUGAGUC]RNA-TT[DNA] (SEQ ID NO.16) Control: 5′[GAUAGCAAUGACGAAUGCGUA]RNA-TT[DNA] (SEQ ID NO.17) 5′[UACGCAUUCGUCAUUGCUAUC]RNA-TT[DNA] (SEQ ID NO.18) LNCap: 5′[GCAUCAGUUCGCUUUUGAC]RNA-TT[DNA] (SEQ ID NO.19) 5′[GUCAAAAGCGAACUGAUGC]RNA-TT[DNA] (SEQ ID NO.20)

Multiple subclones of the human prostate carcinoma line LNCaP were used in this study. The original line, LNCaP, is androgen dependent. The cells LN70, obtained by repeated passages of the line LNCaP in vitro have a diminution in their response to androgens. The clone LNS5, obtained after passage of the animals in an animal, is androgen resistant.

III—Results

The LNCaP cells were transfected in vitro with siRNA AR or control siRNAs using the transfection agent Polyfect (Qiagen). 48 h after transfection, the cells were detached from their support. Half of the cells were used for performing a Western blot detection of the androgen receptor; the other half were put back in culture. The androgen receptor (band at 110 kDa) was no longer detectable by Western blot in the cells transfected by siRNA AR (FIG. 4A). The cells transfected by siRNA and put back in culture were found to be incapable of continuing their growth, to the opposite of the cells transfected by the control siRNA.

The level of response to the androgens was measured by transfecting different cellular clones of the lone LNCaP with a reporter vector placing the coding sequence of luciferase downstream of a minimal promoter flanked by 4 repetitions of the androgen response element (4×ARE). After transfection, the cells were incubated for 18 h in the absence of serum and in the presence or absence of a metabolically stable analogue of dihydrotesterone, R1881 (NEN). The ratio of the luciferase activities under these two conditions makes it possible to measure the level of response to the androgens of the reporter vector.

We measured the effect of the cotransfection in the control RNAi or RNAi AR cells on the response to the androgens of the different clones of the line LNCaP.

FIG. 4B shows a complete inhibition of the response to the androgens in the two androgen-sensitive clones: LNCaP and LNCaP p70. This method does not permit measurement of the response of the androgen-resistant clone LNS5 to the treatment by RNAi AR.

The androgen receptor present in the line LNCAP carries a mutation. We used two different siRNAs to inhibit its synthesis, the previously used siRNA AR and siRNA LNCaP specifically recognizing the mutation LNCaP. The response to the androgens was measured as in experiment 4B (FIG. 4C).

In order to study the effect of the inhibition of the expression of the androgen receptor on tumor growth in vivo of the prostate carcinoma cells, carcinoma cells LNCaP, transfected by a control siRNA (group A) or siRNA AR (group B) were injected subcutaneously in male nude mice. Tumor growth was monitored at regular intervals. It was seen that the tumors of the group B animals started growing later than those of group A and that the volume of the tumors of group B on the 48th day was markedly smaller than that of the tumors of group A (FIG. 4D).

In another experiment, LNCaP cells were injected in male nude mice. When, on the 34^(th) day, the tumors had reached a volume comprised between 1.2 and 1.5 cm³, the mice received via the intraperitoneal route an injection of 2 μg of control siRNA or siRNA AR in 100 μl of PBS. This injection was repeated on the 40 day. It was seen that the administration of siRNA AR leads to a slowing down of the tumor growth (FIG. 4E).

EXAMPLE 5 Inhibition of the Wild or Mutant Forms of the Protein p53

I—Preparation of the Oligonucleotides

The three siRNAs whose sequences are presented below were prepared, one directed against the wild form of p3 and the other directed against the mutated form expressed in a patient which resulted in the establishment of a line.

This mutation corresponds to one of the three observed most frequently in human tumors:

wild p53: Sense: [ G CAUGAACCGGAGGCCCAU]RNA[TT]DNA (SEQ ID NO.21) Anti: [AUGGGCCUCCGGUUCAUG C ]RNA[TT]DNA (SEQ ID NO.22) p53 MT1 (r248w): Sense: [GCAUGAACUGGAGGCCCAU]RNA[TT]DNA (SEQ ID NO.23) Anti: [AUGGGCCUCCAGUUCAUGC]RNA[TT]DNA (SEQ ID NO.24) p53 MT2 (r248w): Sense: [

CAUGAACUGGAGGCCCAU]RNA[TT]DNA (SEQ ID NO.25) Anti: [AUGGGCCUCCAGUUUCAUGA]RN

[TT]DNA (SEQ ID NO.26)

The underlined nucleotides in the wild p53 are those that mutated in the mutant form and which are in italics in the sequences of the mutated form of mutated p53 (p53 MT1 and MT2). The bases in bold above are mutations which were introduced in order to augment the specificity.

II—Results

As shown in FIG. 5B, the H1299-NCI cells, which do not express p53, were transfected (using lipofectamine) by expression vectors (400 ng) of wild p53 (WT) or mutated p53 (MT). siRNAs (in increasing doses: 0, 125 ng, 250 ng, 500 ng and 1000 ng) directed against the wild form (WT), the mutated form (MT1 and MT2) or an irrelevant siRNA (C) were transfected at the same time. The cells were collected 24 hours later and analyzed by Western blot with an antibody directed against p53.

As shown in FIG. 5C, the H1299-NCI cells, which did not express p53, were transfected (using lipofectamine) by expression vectors (400 ng) of wild p53 (WT), mutated p53 (MT) or a mixture of the two (WT+MT) as indicated. siRNAs (400 ng) directed against the wild form (WT), the mutated form (MT1) or an irrelevant siRNA (C) were transfected at the same time. The cells were collected 24 hours later and analyzed by Western blot (ib: immunoblot) with cellular actin (Sigma) to monitor the amount of proteins used in the test.

As shown in FIG. 5D, U2OS cells (human osteosarcoma expressing a wild form of p53) were transfected in a stable manner either by a vector expressing a mutant form of p53 (R248W) or by the corresponding empty vector (pCDNA3). These lines were transfected by the indicated siRNAs and the expression of the indicated proteins was detected by Western blot.

In all cases, the siRNA directed against the mutated form of the protein inhibited the mutated form and the siRNA directed against the wild form inhibited the wild form. Furthermore, there was no crossed reaction because the siRNA directed against the wild form had no effect on the mutated form and vice versa. It should be noted that the expression of the mutant stabilizes the wild protein when it is co-expressed. Consequently, the inhibition of the mutant through its indirect effect brings the wild form to its base level without there being any inhibition of the expression of the protein.

As shown in FIG. 5E, the cells used in FIG. 5D were transfected by the indicated siRNAs. The cells were then subjected to a genotoxic stress by treatment with doxorubicin (200 ng/ml) for 24 h. FIG. 5E shows the analysis of the cell cycle of these cells by incorporation of propidium iodine and FACS analysis. The cells not transfected with the mutant form and thus only expressing the wild form (PCDNA cells) exhibited a high percentage of stopping at G1 in the presence of doxorubicin. The treatment of these cells with wild siRNA, diminishing the wild p53, reduced this stopping at G1. The cells expressing the mutated and wild form (R248W) stopped very little at G1 in the presence of doxorubicin, showing that the mutated form inhibits the activity of the wild form. When these cells were treated with siRNA MT1, they recovered a normal capacity (to compare with the untreated PCDNA controls) of stopping at G1, showing the restoration of the wild p53 activity in these cells.

As shown in FIGS. 5 F, G and H, the MDA 087 cells (stemming from a patient suffering from a Li-Fraumeni cancer syndrome and expressing the mutant R248W) were transfected with a siRNA directed against the mutant form (MT1) of p53, or with an irrelevant siRNA (C) (1.6 μg). Expression of p53 was detected in these cells by Western blot (FIG. 5F); the messenger RNAs were measured by quantitative CR (Light Cycler, Roche) (FIG. 5G) or immunofluorescence (FIG. 5H).

The MDA 087 cells were transfected with a siRNA recognizing the wild form (WT) or the mutated form (MT1) of p53 or by a control siRNA then subjected to a genotoxic stress by treatment with doxorubicin (200 ng/ml) for 24 h. The expression of the mutant form of p53 was detected by Western blot in the cells. It can be seen that the cells having received siRNA MT1 were not capable of stabilizing p53 in response to doxorubicin (FIG. 5I).

FIG. 5J shows the effect of the siRNAs MT1 and MT2 in cells that express the wild and mutated forms of p53. H1299-NCI cells, which do not express p53, were transfected (using lipofectamine) by a reporter vector carrying the gene of luciferase under control of a p53 response element and vectors of expression (400 ng) of the wild p53 (WT), mutated p53 (MT) or a mixture of the two (WT+MT), as indicated. siRNAs (400 ng) directed against the wild form (WT), the mutated form (MT1, MT2) or an irrelevant siRNA (C) were transfected at the same time. The cells were collected 24 hours later and analyzed for the expression of luciferase. Only the wild p53 activated the report vector and the co-expression of the mutant form inhibited this activity. The cotransfection of wild siRNA inhibited the expression of the wild protein and thus the residual activation of the reporter gene. The cotransfection of the siRNA MT1 and MT2, in contrast, restored this activation by blocking selectively the expression of the mutated form and preventing the negative transdominant effect that it exerts on the wild form of p53.

FIG. 5K shows a similar result on the expression of one of the targets of p53, the inhibitory protein of cell proliferation p21, in cells treated as in FIG. 5F. The expression of p21, detected by Western blot, was activated by wild p53 and inhibited when the mutant was co-expressed. This inhibition was lifted in the presence of siRNA MT 1.

EXAMPLE 6 Inhibition of the Viral Protein E6

I—Preparation of the Oligonucleotides

A siRNA directed against the HPV protein E6 was also prepared. It responds to the following sequence:

HPV-16-S2 Sense: 5′[CCACAGUUAUGCACAUAUC]RNA[TT]DNA (SEQ ID NO.27) Anti: 5′[GCUCUGUGCAUAACUUGG]RNA[TT]DNA (SEQ ID NO.28) II—Results

As shown in FIG. 6B, CasKi and SiHA cells, both expressing HPV protein E6, were transfected with the indicated siRNAs, treated or not treated as indicated with doxorubicin and analyzed by Western blot using the indicated antibodies. Treatment of the cells with siRNA E6 induced an augmentation in the expression of P53. This expression of p53 was manifested by an augmentation of the expression of the protein p21.

As shown in FIG. 6C, the cell cycle of the treated siHA cells as in FIG. 6B was analyzed by FACS. The figure represents a characteristic experiment. There was seen an augmentation of cells in phase G1 (FIG. 6D) in the cells treated with siRNA E6, an augmentation which was also seen in the cells when they were treated with doxorubicin.

EXAMPLE 7 Effect of the RNA/RNA Oligonucleotides and the DNA/RNA Hybrids

I—Introduction

The invention envisages the use of DNA/RNA hybrid oligonucleotides as alternative to the RNA/RNA oligonucleotides for inhibiting specifically the expression of a gene. In the case of the DNA/RNA hybrids, the sense strand is preferentially of a DNA nature and the antisense strand of a RNA nature. The other aspects related notably to the size of the oligonucleotides, the nature of the 3′ ends and the mode of synthesis are the same as for the RNA/RNA oligonucleotides. The applications of these DNA/RNA hybrids are identical to those previously described for the RNA/RNA siRNA especially with regard to the therapeutic and diagnostic applications and the validation of genes. However, the doses of oligonucleotides employed in order to obtain the same effects with the DNA/RNA hybrids and RNA/RNA can be different.

II—Preparation of the Oligonucleotides

The sense strand is the one whose sequence is identical to that of the messenger RNA. The antisense strand is the strand complementary to the sense strand. By convention, in a duplex the nature of the strands is indicated in the order sense/antisense. Thus, for example, a DNA/RNA hybrid, noted as D/R, is a oligonucleotide the sense strand of which is of a DNA nature and the antisense strand of which is of a RNA nature and of a sequence complementary to the messenger RNA.

In the experiments described, the oligonucleotides whose sequence is indicated below were used.

For the GFP:

GFP: Sense: [GCAAGCTGACCCTGAAGTTCAT]DNA (SEQ ID NO.29) Anti: [GAACUUCAGGGUCAGCUUGCCG]RNA (SEQ ID NO.30) Control GFP: Sense: [CAUGUCAUGUGUCACAUCUC]RNA[TT]DNA (SEQ ID NO.31) Antisense: [GAGAUGUGACACAUGACAUG]RNA[TT]DNA (SEQ ID NO.32)

FOR THE LNCaP: The underlined bases below correspond to the mutation of the androgen receptor expressed in the cells of human prostate carcinoma (LNCaP).

LNCaP: Sense: [GCATCAGTTCGCTTTTGACTT]DNA (SEQ ID NO.33) [GCAUCAGUUCGCUUUUGAC]RNA-TT[DNA] (SEQ ID NO.34) Antisense: [GTCAAAAGCGAACTGATGCTT]DNA (SEQ ID NO.35) [GUCAAAAGCGAACUGAUGC]RNA = TT[DNA] (SEQ ID NO.36) Control LNCaP: Sense: [GUUCGGUCUGCUUACACUA]RNA-TT[DNA] (SEQ ID NO.37) Antisense: [UAGUGUAAGCAGACCGAAC]RNA-TT[DNA] (SEQ ID NO.38)

For p53:

-   -   The DNA of the hybrids noted H1 comprise RNA bases (U,         underlined).     -   The mutation present in the MT1 oligonucleotides is indicated in         italics.

WT: Sense: 5′[GCAUGAACCGGAGGCCCAU]RNA[TT]DNA (SEQ ID NO.39) Anti: 5′[AUGGGCCUCCGGUUCAUGC]RNA[TT]DNA (SEQ ID NO.40) WT H1 D/R: Sense: 5′[GCA U GAACCGGAGGCCCA U TT]DNA (SEQ ID NO.41) Anti: 5′[AUGGGCCUCCGGUUCAUGC]RNA[TT]DNA (SEQ ID NO.42) WT H1 R/D: Sense: 5′[GCAUGAACCGGAGGCCCAU]RNA[TT]DNA (SEQ ID NO.43) Anti: 5′[A U GGGCC U CCGG UU CA U GCTT]DNA (SEQ ID NO.44) WT H2 R/D: Sense: 5′[GCATGAACCGGAGGCCCATTT]DNA (SEQ ID NO.45) Anti: 5′[AUGGGCCYCCGGUUCAYGC]RNA[TT]DNA (SEQ ID NO.46) WT H2 R/D: Sense: 5′[GCAUGAACCGGAGGCCCAU]RNA[TT]DNA (SEQ ID NO.47) Anti: 5′[ATGGGCCUTCCGGTTCATGCTT]DNA (SEQ ID NO.48) MT1 (R248W)**: Sense: 5′[GCAUGAACUGGAGGCCCAU]RNA[TT]DNA (SEQ ID NO.49) Anti: 5′[AUGGGCCUCC

GUUCAUGC]RNA[TT]DNA (SEQ ID NO.50) MT1 H1 D/R: Sense: 5′[GCA U GAAC U GGAGGCCCA U TT]DNA (SEQ ID NO.51) Anti: 5′[AUGGGCCUCC

GUUCAUGC]RNA[TT]DNA (SEQ ID NO.52) MT1 H1 R/D: Sense: 5′[GCAUGAACUGGAGGCCCAU]RNA[TT]DNA (SEQ ID NO.53) Anti: 5′[A U GGGCC U CC

G UU CA U GCTT]DNA (SEQ ID NO.54) MT1 H2 D/R: Sense: 5′[GCATGAAC

GGAGGCCCATTT]DNA (SEQ ID NO.55) Anti: 5′[AUGGGCCUCC

GUUCAUGC]RNA[TT]DNA (SEQ ID NO.56) MT1 H2 R/D: Sense: 5′[GCATGAAC

GGAGGCCCAT]RNA[TT]DNA (SEQ ID NO.57) Anti: 5′[AUGGGCCUCC

GUUCAUGCTT]DNA (SEQ ID NO.58) II—Results 1) Inhibition of the GFP (Green Fluorescent Protein) by the DNA/RNA Hybrids

The control siRNAs (R/R) or GFP (D/R) in increasing doses were introduced by transfection using the Polyfect kit in C2C12 mouse myoblasts at the same time as a GFP expression vector. The GFP level was monitored by Western blot (FIG. 7A) and by direct measurement of the fluorescence emitted by the GFP by means of a fluorometer (FIG. 7B). There was seen a strong inhibition (up to 80%) of the expression of GFP by the DNA/RNA hybrid siRNAs.

2) Inhibition of the Androgen Receptor by the DNA/RNA Hybrids

FIG. 7D shows that the H1 D/R hybrids are as effective as the R/R for inhibiting the expression of genes. H1299-NCI cells, which do not express p53, were transfected (using lipofectamine) by vectors of expression (400 ng) of wild p53 (WT), mutated p53 (MT) or a mixture of the two (WT+MT), as indicated. A CMV-GFP vector was also transfected as internal control. The siRNAs (400 ng) directed against the wild form (WT), the mutated form (MT) or an irrelevant siRNA (CTRL) were transfected at the same time. The cells were collected 24 hours later and analyzed by Western blot with an antibody directed against p53 (D01, Santa Cruz) or GFP (Santa-Cruz) to monitor the transfection efficacy. Note: the expression of the mutated form of the protein stabilizes the wild form.

FIG. 7E shows that the H2 D/R hybrids were as effective as the R/R for inhibiting the expression of the genes. The H1299-NCI cells, which do not express p53, were transfected (using lipofectamine) by expression vectors (400 ng) of wild p53 (WT), mutated p53 (MT) and a mixture of the two (WT+MT) as indicated. The siRNAs (400 ng) directed against the wild form (WT), the mutated form (MT) or an irrelevant siRNA (C) were transfected at the same time. The cells were collected 24 hours later and analyzed by Western blot with an antibody directed against p53 (D01, Santa Cruz).

EXAMPLE 8 Administration In Vivo of siRNA Via Different Routes

Tumor cells expressing luciferase in a stable manner were injected subcutaneously to nude mice (1 million cells in the right flank). On the 8^(th) day of tumor growth, the tumors having an average volume of 200 mm³ were injected either with control siRNAs (mixed sequence of HIF1α, see example 3) or with a siRNA directed against luciferase. The control siRNAs (3 μg/mouse) were injected in a volume of 50 μl in PBS via the subcutaneous route in the animal's flank.

The luciferase siRNAs were injected at the rate of 3 μg/mouse (3 animals in each group) in 50 μl of PBS via the subcutaneous route (sc), the intraperitoneal route (ip), the intravenous route (iv) (tail vein) or the intratumoral route (it). In this latter case, the luciferase siRNAs (3 μg/mouse) were diluted in only 20 μl of PBS.

Three days after injection of the siRNAs, the animals were sacrificed, the tumors were collected and homogenized with a Polytron grinder. Quantitative determination of the proteins and measurement of the luciferase activity in a luminometer were performed on the homogenates.

The results shown in FIG. 8 show the luciferase activity in relation to the quantity of protein. 

1. A method for reducing tumor volume of prostate cancer and fibrosarcoma in a subject, comprising administering to said subject a pharmaceutical composition comprising a double-strand oligonucleotide consisting of SEQ ID NO: 7 and SEQ ID NO:
 8. 2. A method for treating a pathological hypervascularization in tumoral development wherein VEGF is overexpressed, comprising directly contacting a VEGF overexpressing tumor cell with a pharmaceutical composition comprising a double-strand oligonucleotide consisting of SEQ ID NO: 7 and SEQ ID NO:
 8. 3. A method for inhibiting VEGF expression in a subject, comprising administering to said subject a pharmaceutical composition comprising a double-strand oligonucleotide consisting of SEQ ID NO: 7 and SEQ ID NO:
 8. 